Single-molecule tracking (SMT) in live cells reveals how biomolecules explore crowded intracellular environments, yet most tracking software assumes Brownian motion, an approximation that fails when anomalous diffusion dominates. This leads to biased trajectory reconstruction and loss of biophysical information, particularly for the short trajectories typical of intracellular experiments. We present FreeTrace, an SMT framework that reconstructs trajectories under fractional Brownian motion (fBm), incorporating temporal correlations directly into linking with minimal input parameters. A deep neural network estimates diffusion properties (Hurst exponent H and generalised diffusion coefficient K ) for individual trajectories, while an analytical ensemble estimator accurately recovers H from trajectories as short as three frames, conditions where mean-squared displacement methods fail. Benchmarking on simulated data demonstrates superior performance across motion types and densities. Applications to chromatin-bound histones, DNA repair proteins in S.c. yeast and human cells reveal biologically meaningful diffusion subpopulations, with H values consistent with polymer models and confined motion. FreeTrace bridges theoretical anomalous diffusion models and routine biological experiments. ### Competing Interest Statement The authors have declared no competing interest.

Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open up new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity as well as axial calibration issues. In this work, we address this issue by leveraging the geometrical characteristics of fractal-like structures, which serve as axial calibration tools and modulate cell growth. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as an alternative to electron microscopy for material imaging but also as calibration tools for 3D super-resolution microscopy. We used their multiscale and known geometry to perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. We then utilized these substrates as a platform for high-resolution bioimaging. As a proof of concept, we cultivate human mesenchymal stem cells on these substrates, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with a 3D SMLM assessment.

In recent years, the segmentation of short molecular trajectories with varying diffusive properties has drawn particular attention of researchers, since it allows studying the dynamics of a particle. In the past decade, machine learning methods have shown highly promising results, also in changepoint detection and segmentation tasks. Here, we introduce a novel iterative method to identify the changepoints in a molecular trajectory, i.e. frames, where the diffusive behavior of a particle changes. A trajectory in our case follows a fractional Brownian motion and we estimate the diffusive properties of the trajectories. The proposed Bottom-up iterative anomalous diffusion detector (BI-ADD) combines unsupervised and supervised learning methods to detect the changepoints. Our approach can be used for the analysis of molecular trajectories at the individual level and also be extended to multiple particle tracking, which is an important challenge in fundamental biology. We validated BI-ADD in various scenarios within the framework of the 2nd anomalous diffusion challenge 2024 dedicated to single particle tracking. Our method is implemented in Python and is publicly available for research purposes.

Controlling quantum light-matter interactions at scales smaller than the diffraction limit at the single quantum emitter level is a critical challenge to the goal of advancing quantum technologies. We introduce a novel material platform that enables precise engineering of spontaneous emission changes in molecular single emitters through 3D nanofields. This platform is based on a 3D hollow plasmonic nanomaterial arranged in a square lattice, uniformly scalable to the centimeter scale while maintaining unit cell geometry. This coupled system leads to billions of Purcell-enhanced single emitters integrated into a nanodevice. Using far-field single-molecule super-resolution microscopy, we investigate emission modifications at the single-emitter level, enabling molecular position sensing with resolution surpassing the diffraction limit. By combining the nanolocalization with time correlation single photon counting, we probe molecule per molecule enhanced quantum light-matter interactions. This 3D plasmonic geometry significantly enhances light-matter interactions, revealing a broad range of lifetimes – from nanoseconds to picoseconds – significantly increasing the local density of states in a manner that depends on both molecular position and dipole orientation, offering extreme position sensitivity within the 3D electromagnetic landscape. By leveraging these plasmonic nanostructures and our method for measuring single-molecule Purcell-enhanced nano-resolved maps, we enable fine-tuned control of light-matter interactions. This approach enables the on-demand control of fast single-photon sources at room temperature, providing a powerful tool for molecular sensing and quantum applications at the single-emitter level.

High refractive index dielectric nanoantennas strongly modify the decay rate via the Purcell effect through the design of radiative channels. Due to their dielectric nature, the field is mainly confined inside the nanostructure and in the gap, which is hard to probe with scanning probe techniques. Here we use single-molecule fluorescence lifetime imaging microscopy (smFLIM) to map the decay rate enhancement in dielectric GaP nanoantenna dimers with a median localization precision of 14 nm. We measure, in the gap of the nanoantenna, decay rates that are almost 30 times larger than on a glass substrate. By comparing experimental results with numerical simulations we show that this large enhancement is essentially radiative, contrary to the case of plasmonic nanoantennas, and therefore has great potential for applications such as quantum optics and biosensing.

Event-based sensors (EBS), or neuromorphic vision sensors, offer a novel approach to imaging by recording light intensity changes asynchronously, unlike conventional cameras that capture light over fixed exposure times. This capability results in high temporal resolution, reduced data redundancy, and a wide dynamic range. This makes EBS ideal for Single-Molecule Localization Microscopy (SMLM) as SMLM relies on the sequential imaging of sparse, blinking fluorescent emitters to achieve super-resolution. Recent studies have shown that EBS can effectively capture these emitters, achieving spatial resolution comparable to traditional cameras. However, existing analyses of event-based SMLM (eveSMLM) data have relied on converting event lists into image frames for conventional analysis, limiting the full potential of the technology. To overcome this limitation, we developed EVE, a specialized software for analyzing eveSMLM data. EVE offers an integrated platform for detection, localization, and post-processing, with various algorithmic options tailored for the unique structure of eveSMLM data. EVE is user-friendly and features an open, modular infrastructure that supports ongoing development and optimization. EVE is the first dedicated tool for event-based SMLM, transforming the analysis process to fully utilize the spatiotemporal data generated by EBS. This allows researchers to explore the full potential of eveSMLM and encourages the development of new analytical methods and experimental improvements. ### Competing Interest Statement The authors have declared no competing interest.

One of the major challenges in nanophotonics is the direct measurement of the interaction of a nanostructure with a single fluorescent emitter at the nanometer level. Several approaches developed to this aim can be found in the literature. Recently, single molecule localization imaging, traditionally used for the study of biological samples, entered the nanophotonics realm opening new horizons. We report on the development of single-molecule fluorescence lifetime imaging microscopy (sm-FLIM), an innovative approach enabling the simultaneous measurement of the lifetime and the intensity of single-molecules densely labeling a nanostructured sample, with a field of view of 10 µm2 , a spatial resolution of approximately 14 nm and a temporal resolution of approximately 50 ps. smFLIM enabled us to image, at the single molecule level, the Local Density of Optical States (LDOS, which is related to the inverse of the fluorescence lifetime) of dielectric nanoantennas and periodic arrays of hollow truncated plasmonic nanocones.

3D ceramic architectures are captivating geometrical features with an immense demand in optics. In this work, an additive manufacturing (AM) approach for printing alkaline-earth perovskite 3D microarchitectures is developed. The approach enables custom-made photoresists suited for two-photon lithography, permitting the production of alkaline-earth perovskite (BaZrO3 , CaZrO3 , and SrZrO3 ) 3D structures shaped in the form of octet-truss lattices, gyroids, or inspired architectures like sodalite zeolite, and C60 buckyballs with micrometric and nanometric feature sizes. Alkaline-earth perovskite morphological, structural, and chemical characteristics are studied. The optical properties of such perovskite architectures are investigated using cathodoluminescence and wide-field photoluminescence emission to estimate the lifetime rate and defects in BaZrO3 , CaZrO3 , and SrZrO3 . From a broad perspective, this AM methodology facilitates the production of 3D-structured mixed oxides. These findings are the first steps toward dimensionally refined high-refractive-index ceramics for micro-optics and other terrains like (photo/electro)catalysis.

At Langevin Institute, ESPCI Paris, we have developed a label-free approach to detect and track viruses in aquatic solutions using an interferometric microscopy approach working in transmission. By improving the experimental setup design, we have increased the sensitivity to detect viruses as small as 20nm in diameter, AAV viruses, which are of paramount importance for gene therapy. Furthermore, based on the variation of the interferometric signal along the axial position, we introduce a new way to perform single-shot volumetric imaging which enables high-precision 3D tracking.

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments.

Single-molecule localization microscopy (SMLM) is often hampered by the fixed frame rate of the acquisition. Here, we present an alternative new approach to data acquisition and processing based on an affordable event-based sensor. This type of sensor reacts to light intensity changes rather than integrating photons during each frame exposure time. This makes it particularly suited to SMLM, where the ability to surpass the diffraction-limited resolution is provided by blinking events. Each pixel works independently and returns a signal only when an intensity change is detected. Since the output is a list containing only useful data rather than a series of frames, the temporal resolution is significantly better than typical scientific cameras. We demonstrate event-based SMLM super-resolution imaging on biological samples with spatial resolution on par with EMCCD or sCMOS performance. Furthermore, taking advantage of its unique properties, we use event-based SMLM to perform very dense single-molecule imaging, where framebased cameras experience significant limitations.

Viruses have a profound influence on all forms of life, motivating the development of rapid and minimally invasive methods for virus detection. In this study, we present a novel methodology that enables quantitative measurement of the interaction between individual biotic nanoparticles and antibodies in solution. Our approach employs a label-free, full-field common-path interferometric technique to detect and track biotic nanoparticles and their interactions with antibodies. It is based on the interferometric detection of light scattered by viruses in aqueous samples for the detection of individual viruses.We employ single-particle tracking analysis to characterize the size and properties of the detected nanoparticles, and to monitor the changes in their diffusive mobility resulting from interactions. To validate the sensitivity of our detection approach, we distinguish between particles having identical diffusion coefficients but different scattering signals, using DNA-loaded and DNA-devoid capsids of the Escherichia coli T5 virus phage.In addition, we have been able to monitor, in real time, the interaction between the bacteriophage T5 and purified antibodies targeting its major capsid protein pb8, as well as between the phage SPP1 and nonpurified anti-SPP1 antibodies present in rabbit serum. Interestingly, these virus-antibody interactions are observed within minutes. Finally, by estimating the number of viral particles interacting with antibodies at different concentrations, we successfully quantify the dissociation constant Kd of the virus-antibody reaction using single-particle tracking analysis.

The potential of micro-and nanofabricated samples as a platform to modulate cell behavior using 3D physical cues has shown tremendous interest in cell differentiation. Investigating cell behavior and organization at the molecular level requires advanced imaging techniques. We produced multiscale 3D substrates with glass fractal pyramids composed of several generations of octahedra of decreasing sizes, which allow direct observation of cells in 3D SMLM. We show how these samples can be fluorescently labeled and used as a self-referenced sample for calibration and resolution measurements. Moreover, we perform quantitative 3D SMLM on cells growing on such fractal substrates, observing spheroid-like behavior.

Digital holography is an imaging technique that enables a 3-dimensional reconstruction of the electromagnetic field scattered by an object in both amplitude and phase. We demonstrated its use in microscopy in linear regime for the full 3-D mapping of the field scattered by single nanostructures such as nano-antennas [1] and near-field probes [2]. Holography is a technique based on interferences, which can be obtained at the laser illumination wavelength, but also with Second Harmonic Generation (SHG), since the latter is produced in a coherent process [3, 4]. Here, we describe the development of a harmonic holographic microscope for single-shot mapping of the second harmonic 3D radiation pattern near samples with nonzero second harmonic susceptibilities. The knowledge of the scattered field (amplitude and phase) in a given plane (that of the camera) allows its reconstruction in any other plane using e.g. the angular spectrum representation of the optical fields [5], and assuming propagation in homogeneous media, a process called 3D numerical back-propagation [6]. In addition to providing 3D reconstruction, thus enhancing the imaging capabilities beyond those of back focal-plane imaging, the harmonic holography microscope also benefits from an amplification effect since the signal from the sample is multiplied by an intense reference in the interference term [7], making the method particularly well suited to measure the weak SHG signals [8]. After a first validation on dielectric samples made of nonlinear micro-crystals and cornea collagen, we are implementing the technique to obtain SHG fields radiated by plasmonic nano-antennas [9].

In this contribution, we will report on a novel technique recently developed at Institut Langevin that enables multiplexed and super-resolved fluorescence lifetime imaging at the single-molecule level (smFLIM) with a field of view of ~10 µm2 and a localization precision of ~15 nm. Our method combines the use of an EMCCD camera for the localization of photoactivatable single molecules (as usually done in SMLM) and a linear array of single-photon avalanche diodes (SPADs) for time-resolved measurements. Our method can be used for LDOS imaging of disordered and deterministic structures with nanometer-sized features and extensions going from a few hundred nanometers to several microns, with a dynamic temporal range spanning from ns to ms. We will show how smFLIM allows the study of simple nanostructures such as a silver nanowire and complex nanostructures, such as periodic arrays of hollow gold truncated nanocones (nanochimneys).

La nanophotonique s’intéresse à la génération et au contrôle de la lumière grâce à la nanostructuration de la matière, ainsi qu’à l’imagerie de ces nanostructures. Mais comment étudier les propriétés optiques de ces matériaux à l’échelle nanométrique ? Dans cet article, nous présentons une nouvelle technique inspirée d’approches de microscopie à super-résolution établies en bioimagerie que nous avons adaptée au domaine de la nanophotonique dans le but d’obtenir des cartographies super-résolues de l’interaction lumière-matière.

Long wave infrared (LWIR) radiation (7-14 μm) allows illumination-less imaging, and spectroscopic chemical identification. Infrared imaging is ubiquitous in defense thermography, airborne and atmospheric sensing, fault detection, and medical testing. Visible speckle imaging can successfully image through complex scattering media. We describe a novel broadband LWIR speckle imaging-based wavefront sensor, utilizing a thin diffuser with an uncooled microbolometric camera. Due to the thin diffuser, local phase gradients produce speckle deformations which are estimated by a rapid image registration algorithm to generate a phase gradient map, whose 2-D integration yields the reconstructed wavefront. We demonstrate LWIR wavefront reconstruction using our setup in infrared optical samples, with future applications for LWIR imaging through visually non-transparent materials.

In article number 2102758, Jedrzej Winczewski, Arturo Susarrey Arce, Han Gardeniers and co-workers present additively-manufactured 3D luminescent microstructures emitting light in the visible range. The 3D europium-doped zirconia (ZrO2:Eu3+) architectures are fabricated via two-photon lithography using custom-made photoresin. The orange-red emission of ZrO2:Eu3+ microstructures of sub-micrometer feature size definition is demonstrated using fluorescence microscopy and investigated in detail with cathodoluminescence. The presented structuring technology provides a new platform for developing 3D luminescent microdevices and optimizing phosphor-based light emitters further.

A novel broadband infrared (IR) speckle imaging system with a thin scatterer and an uncooled microbolometric camera is employed to encode wavefront phase variations as local speckle deformations. The phase reconstruction from speckle shifts using a fast diffeomorphic algorithm ultimately demonstrates IR wavefront reconstruction through complex media.

Implementation of more refined structures at the nano to microscale is expected to advance applications in optics and photonics. This work presents the additive manufacturing of 3D luminescent microarchitectures emitting light in the visible range. A tailor‐made organo‐metallic resin suitable for two‐photon lithography is developed, which upon thermal treatment in an oxygen‐rich atmosphere allows the creation of silicon‐free tetragonal (t‐) and monoclinic (m‐) ZrO2. The approach is unique because the tailor‐made Zr‐resin is different from what is achieved in other reported approaches based on sol−gel resins. The Zr‐resin is compatible with the Eu‐rich dopant, a luminescent activator, which enables to tune the optical properties of the ZrO2 structures upon annealing. The emission characteristics of the Eu‐doped ZrO2 microstructures are investigated in detail with cathodoluminescence and compared with the intrinsic optical properties of the ZrO2. The hosted Eu has an orange−red emission showcased using fluorescence microscopy. The presented structuring technology provides a new platform for the future development of 3D luminescent devices.