Ultrafast ultrasound localization microscopy can be used to detect the subwavelength acoustic scattering of intravenously injected microbubbles to obtain haemodynamic maps of the vasculature of animals and humans. The quality of the haemodynamic maps depends on signal-to-noise ratios and on the algorithms used for the localization of the microbubbles and the rendering of their trajectories. Here we report the results of benchmarking of the performance of seven microbubble-localization algorithms. We used metrics for localization errors, localization success rates, processing times and a measure of the reprojection of the localization of the microbubbles on the original beamformed grid. We combined eleven metrics into an overall score and tested the algorithms in three simulated microcirculation datasets, and in angiography datasets of the brain of a live rat after craniotomy, an excised rat kidney and a mammary tumour in a live mouse. The algorithms, metrics and datasets, which we have made openly available at https://github.com/AChavignon/PALA and https://doi.org/10.5281/zenodo.4343435 , will facilitate the identification or generation of optimal microbubble-localization algorithms for specific applications.

Datasets provided for Open Platform for Ultrasound Localization Microscopy: Performance Assessment of Localization Algorithms. Abstract: Ultrasound Localization Microscopy (ULM) is an ultrasound imaging technique that relies on the acoustic response of sub-wavelength ultrasound scatterers to map the microcirculation with an order of magnitude increase in resolution. Initially demonstrated in vitro, this technique has matured and sees implementation in vivo for vascular imaging of organs, and tumors in both animal models and humans. The performance of the localization algorithm greatly defines the quality of vascular mapping. We compiled and implemented a collection of ultrasound localization algorithms and devised three datasets in silico and in vivo to compare their performance through 18 metrics. We also present two novel algorithms designed to increase speed and performance. By openly providing a complete package to perform ULM with the algorithms, the datasets used, and the metrics, we aim to give researchers a tool to identify the optimal localization algorithm for their usage, benchmark their software and enhance the overall image quality in the field while uncovering its limits. This article provides all materials and post-processing scripts and functions. Article to be cited: Heiles, Chavignon, Hingot, Lopez, Teston and Couture.Performance benchmarking of microbubble-localization algorithms for ultrasound localization microscopy, Nature Biomedical Engineering, 2022, (doi.org/10.1038/s41551-021-00824-8). Related processing scripts and codes: github.com/AChavignon/PALA Request on data: arthur.chavignon.pro(at)gmail.com

Ultrasound-vaporizable microdroplets can be exploited for targeted drug delivery. However, it requires customized microfluidic techniques able to produce monodisperse, capillary-sized and biocompatible multiple emulsions. Recent development of microfluidic devices led to the optimization of microdroplet production with high yields, low polydispersity and well-defined diameters. So far, only few were shown to be efficient for simple droplets or multiple emulsions production below 5 μm in diameter, which is required to prevent microembolism after intravenous injection. Here, we present a versatile microchip for both simple and multiple emulsion production. This parallelized system based on microchannel emulsification was designed to produce perfluorocarbon in water or water within perfluorocarbon in water emulsions with capillary sizes (<5 μm) and polydispersity index down to 5% for in vivo applications such as spatiotemporally-triggered drug delivery using Ultrasound. We show that droplet production at this scale is mainly controlled by interfacial tension forces, how capillary and viscosity ratios influence droplet characteristics and how different production regimes may take place. The better understanding of droplet formation and its relation to applied pressures is supported by observations with a high-speed camera. Compared to previous microchips, this device opens perspectives to produce injectable and biocompatible droplets with a reasonable yield in order to realize preclinical studies in mice.

Once injected into a living organism, cells diffuse or migrate around the initial injection point and become impossible to be visualized and tracked in vivo. The present work concerns the development of a new technique for therapeutic cell labeling and subsequent in vivo visualization and magnetic retention. It is hypothesized and subsequently demonstrated that nanohybrids made of persistent luminescence nanoparticles and ultrasmall superparamagnetic iron oxide nanoparticles incorporated into a silica matrix can be used as an effective nanoplatform to label therapeutic cells in a nontoxic way in order to dynamically track them in real-time in vitro and in living mice. As a proof-of-concept, it is shown that once injected, these labeled cells can be visualized and attracted in vivo using a magnet. This first step suggests that these nanohybrids represent efficient multifunctional nanoprobes for further imaging guided cell therapies development.

Persistent luminescence nanoparticles have recently been proposed as innovative optical probes for small animal in vivo imaging. The main advantage of such probes is their ability to emit light for a long time after the end of their excitation, allowing in vivo imaging with low background. This work reports new information on the physico-chemical characterizations of Cr doped ZnGa2O4 nanoprobes in terms of synthetic procedure, luminescence properties as well as colloidal stabilities in different aqueous media and over the time.

The analysis of the optical spectroscopy of the Cr3+ doped spinel was initiated by Prof. Georges Boulon more than twenty years ago. More recently persistent luminescence nanoparticles of Cr doped zinc gallate have found interest for in vivo imaging of small animals. Here we evaluated near infra-red (NIR) excitation (or NIR photostimulation) via photo-transfer mechanism as an additional tool for in vivo optical imaging. Investigation of the persistent luminescence induced by NIR photostimulation is studied after either a primary UV (band-to-band excitation) or visible irradiation (direct Cr 3d-3d excitation). UV or visible pre-excited ZnGa2O4:Cr (ZGO:Cr) nanoparticles are kept active during several days thanks to deep traps (with depths 1 eV -1.2 eV) observed in these samples which can be probed through thermally stimulated luminescence (TSL) technique showing glow curve maximums at 470 K and 530 K upon visible light excitation. These deep traps are stable at room temperature but can be emptied by NIR light photostimulation. Experiments were carried out to study the photostimulation induced trapping-detrapping in the ZGO:Cr phosphor. Photostimulation was also tested in vivo for small animal optical imaging to offer new perspectives and modalities. (C) 2016 Elsevier B.V. All rights reserved.

Imaging nanoprobes are a group of nanosized agents developed for providing improved contrast for bioimaging. Among various imaging probes, optical sensors capable of following biological events or progresses at the cellular and molecular levels are actually actively developed for early detection, accurate diagnosis, and monitoring of the treatment of diseases. The optical activities of nanoprobes can be tuned on demand by chemists by engineering their composition, size and surface nature. This review will focus on researches devoted to the conception of nanoprobes with particular optical properties, called persistent luminescence, and their use as new powerful bioimaging agents in preclinical assays.

La thérapie cellulaire consiste à utiliser des cellules comme médicament injectable dans le but de favoriser la réparation d'un tissu ou d'un organe. Certaines cellules possèdent la propriété de stimuler la formation de nouveaux vaisseaux sanguins. Elles présentent un intérêt pour le développement d'un traitement des ischémies des membres inférieurs ou du myocarde. Déterminer le devenir de ces cellules après injection in vivo est important pour comprendre les mécanismes responsables de l'efficacité d'un tel traitement. Cependant, il est difficile à observer et très peu décrit dans la littérature. Quelques exemples d'utilisation de nanotechnologies sont rapportés pour suivre des cellules in vitro. Mais des facteurs limitant tels qu'une complexité de mise en oeuvre de ces techniques ou leur trop faible sensibilité rend difficile leur utilisation in vivo. Ce travail de thèse propose de décrire le développement de nanoparticules originales associant les modalités d'imagerie optique et d'IRM afin de marquer simplement des cellules ayant un potentiel thérapeutique. Les protocoles développés ont permis de vectoriser et suivre en temps réel ces cellules après injection chez la souris.

With the fast development of noninvasive diagnosis, the design of multimodal imaging probes has become a promising challenge. If many monofunctional nanocarriers have already proven their efficiency, only few multifunctional nanoprobes have been able to combine the advantages of diverse imaging modalities. An innovative nanoprobe called mesoporous persistent luminescence magnetic nanohybrids (MPNHs) is described that shows both optical and magnetic resonance imaging (MRI) properties intended for in vivo multimodal imaging in small animals. MPNHs are based on the assembly of chromium-doped zinc gallate oxide and ultrasmall superparamagnetic iron oxide nanoparticles embedded in a mesoporous silica shell. MPNHs combine the optical advantages of persistent luminescence, such as real time imaging with highly sensitive and photostable detection, and MRI negative contrast properties that ensure in vivo imaging with rather high spatial resolution. In addition to their imaging capabilities, these MPNHs can be motioned in vitro with a magnet, which opens multiple perspectives in magnetic vectorization and cell therapy research.

We presently introduce a novel generation of optical nanoprobes, based on chromium-doped zinc gallate, whose persistent luminescence can be activated in vivo through living tissues using highly penetrating low energy photons from the red region of the visible spectrum. Surface functionalization of this photonic nanoprobe can be adjusted to favor multiple challenging biomedical applications.

Persistent luminescence is a singular property of some materials which are able to store the excitation or light irradiation energy at intrinsic traps or defects before slowly emitting lower energy photons within several hours. When such compounds are prepared as nanoparticles (NPs), when functionalization is realized to get colloidal materials well dispersed in aqueous medium, such nanoprobes open the use of the persistent luminescence for bioimaging applications. Recently, the numbers of in vivo applications increased with new modalities and new expectations.In this review, we focused our attention on the ZnGa2O4:Cr (ZGO:Cr) nanoparticles. When ZnGa2O4 (ZGO), a normal spinel is doped with Cr3+ ions, a high brightness persistent luminescence material with an emission spectrum perfectly matching the transparency window of living tissues is obtained. It allows in vivo mouse imaging with an excellent target-to-background ratio. One interesting characteristic of ZGO:Cr lies in the fact that its persistent luminescence can be excited with orange/red light, well below its band gap energy and in the transparency window of living tissues. This important property allows multiple re-excitations to perform long term bioimaging. Antisite defects of the direct spinel structure are assumed to provide shallow traps which store the excitation light. Charge release by room temperature thermal excitation and recombination center, here trivalent chromium, are responsible for the persistent luminescence. Following a primary excitation (UV or visible), one also observed that trapped charges can be released under 977 nm light stimulation for several spinel gallate materials, therefore increasing the modalities and the materials envisioned for in vivo excitation of these NPs. (C) 2015 Elsevier B.V. All rights reserved.

Ultra-small ZnGa2 O4 :Cr(3+) nanoparticles (6 nm) that exhibit near-infrared (NIR) persistent luminescence properties are synthesized by using a non-aqueous sol-gel method assisted by microwave irradiation. The nanoparticles are pegylated, leading to highly stable dispersions under physiological conditions. Preliminary in vivo studies show the high potential for these ultra-small ZnGa2 O4 :Cr(3+) nanoparticles to be used as in vivo optical nanotools as they emit without the need for in situ excitation and, thus, avoid the autofluorescence of tissues.

Optical imaging for biological applications requires more sensitive tools. Near-infrared persistent luminescence nanoparticles enable highly sensitive in vivo optical detection and complete avoidance of tissue autofluorescence. However, the actual generation of persistent luminescence nanoparticles necessitates ex vivo activation before systemic administration, which prevents long-term imaging in living animals. Here, we introduce a new generation of optical nanoprobes, based on chromium-doped zinc gallate, whose persistent luminescence can be activated in vivo through living tissues using highly penetrating low-energy red photons. Surface functionalization of this photonic probe can be adjusted to favour multiple biomedical applications such as tumour targeting. Notably, we show that cells can endocytose these nanoparticles in vitro and that, after intravenous injection, we can track labelled cells in vivo and follow their biodistribution by a simple whole animal optical detection, opening new perspectives for cell therapy research and for a variety of diagnosis applications.

ZnGa 2 O 4 (ZGO) is a normal spinel. When doped with Cr 3+ ions, ZGO:Cr becomes a high brightness persistent luminescence material with an emission spectrum perfectly matching the transparency window of living tissues. It allows in vivo mouse imaging with a better signal to background ratio than classical quantum dots. The most interesting characteristic of ZGO:Cr lies in the fact that its LLP can be excited with red light, well below its band gap energy and in the transparency window of living tissues. A mechanism based on the trapping of carriers localized around a special type of Cr 3+ ions namely Cr N2 can explain this singularity. The antisite defects of the structure are the main responsible traps in the persistent luminescence mechanism. When located around Cr 3+ ions, they allow, via Cr 3+ absorption, the storage of not only UV light but also all visible light from the excitation source.

Recently red emitting long-lasting phosphorescence (LLP) materials have been demonstrated to be useful biomarkers for small animal in vivo imaging. We report here our investigations on the optical properties of chromium doped AB(2)O(4) spinels (with A = Zn, Mg and B = Ga, Al) suitable for such applications. It is possible to tune the absorption wavelengths of Cr3+ by a crystal field variation and also slightly vary the emission to be better centered in the biological window and to adjust the trap depth in order to better control the release of the charges. These traps are therefore stable at room temperature and could be emptied by thermal or near infrared source, which makes these materials potential new optically photo-storage compounds. (C) 2014 Elsevier B.V. All rights reserved.

ZnGa2O4 (ZGO) is a normal spinel. When doped with Cr3+ ions, ZGO:Cr becomes a high brightness persistent luminescence material with an emission spectrum perfectly matching the transparency window of living tissues. It allows in vivo mouse imaging with a better signal to background ratio than classical quantum dots. The most interesting characteristic of ZGO:Cr lies in the fact that its LLP can be excited with red light, well below its band gap energy and in the transparency window of living tissues. A mechanism based on the trapping of carriers localized around a special type of Cr3+ ions namely CrN2 can explain this singularity. The antisite defects of the structure are the main responsible traps in the persistent luminescence mechanism. When located around Cr3+ ions, they allow, via Cr3+ absorption, the storage of not only UV light but also all visible light from the excitation source.

A whole organism high-throughput screen of approximately 87,000 compounds against Trypanosoma brucei brucei led to the recent discovery of several novel compound classes with low micromolar activity against this organism and without appreciable cytotoxicity to mammalian cells. Herein we report a structure-activity relationship (SAR) investigation around one of these hit classes, the 3-(oxazolo[4,5-b]pyridin-2-yl)anilides. Sharp SAR is revealed, with our most active compound (5) exhibiting an IC₅₀ of 91 nM against the human pathogenic strain T.b. rhodesiense and being more than 700 times less toxic towards the L6 mammalian cell line. Physicochemical properties are attractive for many compounds in this series. For the most potent representatives, we show that solubility and metabolic stability are key parameters to target during future optimisation.