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Unknown particle screening-including virus and nanoparticles-are keys in medicine, industry, and also in water pollutant determination. Here, RYtov MIcroscopy for Nanoparticles Identification (RYMINI) is introduced, a staining-free, non-invasive, and non-destructive optical approach that is merging holographic label-free 3D tracking with high-sensitivity quantitative phase imaging into a compact optical setup. Dedicated to the identification and then characterization of single nano-object in solution, it is compatible with highly demanding environments, such as level 3 biological laboratories, with high resilience to external source of mechanical and optical noise. Metrological characterization is performed at the level of each single particle on both absorbing and transparent particles as well as on immature and infectious HIV, SARS-CoV-2 and extracellular vesicles in solution. The capability of RYMINI to determine the nature, concentration, size, complex refractive index and mass of each single particle without knowledge or model of the particles' response is demonstrated. The system surpasses 90% accuracy for automatic identification between dielectric/metallic/biological nanoparticles and ≈80% for intraclass chemical determination of metallic and dielectric. It falls down to 50-70% for type determination inside the biological nanoparticle's class.
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Holografia , Nanopartículas Metálicas , Nanopartículas , Vírus , Nanopartículas/química , Microscopia/métodosRESUMO
We report the modification of a label-free image scanning microscope (ISM) to perform asynchronous 2D imaging at up to 24kHz while keeping the lateral resolution gain and background rejection of a regular label-free ISM setup. Our method uses a resonant mirror oscillating at 12kHz for one-direction scanning and a chromatic line for instantaneous scanning in the other direction. We adapt optical photon reassignment in this scanning regime to perform fully optical super-resolution imaging. We exploit the kHz imaging capabilities of this confocal imaging system for single nanoparticle tracking down to 20â nm for gold and 50â nm for silica particles as well as imaging freely moving Lactobacillus with improved resolution.
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We report on the use of a thin diffuser placed in the close vicinity of a camera sensor as a simple and effective way to superlocalize plasmonic nanoparticles in 3D. This method is based on holographic reconstruction via quantitative phase and intensity measurements of a light field after its interaction with nanoparticles. We experimentally demonstrate that this thin diffuser can be used as a simple add-on to a standard bright-field microscope to allow the localization of 100â nm gold nanoparticles at video rate with nanometer precision (1.3â nm laterally and 6.3â nm longitudinally). We exemplify the approach by revealing the dynamic Brownian trajectory of a gold nanoparticle trapped in various pockets within an agarose gel. The proposed method provides a simple but highly performant way to track nanoparticles in 3D.
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Localization microscopy approaches with enhanced depth-of-field (EDoF) are commonly optimized using the Cramér-Rao bound (CRB) as a criterion. It is widely believed that the CRB can be attained in practice by using the maximum-likelihood estimator (MLE). This is, however, an approximation, of which we define in this paper the precise domain of validity. Exploring a wide range of settings and noise levels, we show that the MLE is efficient when the signal-to-noise ratio (SNR) is such that the localization standard deviation of a single molecule is less than 20 nm. Thus, our results provide an explicit and quantitative validity boundary for the use of the MLE in EDoF localization microscopy setups optimized with the CRB.
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Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.
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Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Microscopia de Interferência/métodos , Imagem Individual de Molécula/métodos , Actinas/química , Actinas/fisiologia , Humanos , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/fisiologia , Células-Tronco PluripotentesRESUMO
Single-molecule localization microscopy has become a prominent approach to study structural and dynamic arrangements of nanometric objects well beyond the diffraction limit. To maximize localization precision, high numerical aperture objectives must be used; however, this inherently strongly limits the depth-of-field (DoF) of the microscope images. In this work, we present a framework inspired by "optical co-design" to optimize and benchmark phase masks, which, when placed in the exit pupil of the microscope objective, can extend the DoF in the realistic context of single fluorescent molecule detection. Using the Cramér-Rao bound (CRB) on localization accuracy as a criterion, we optimize annular binary phase masks for various DoF ranges, compare them to Incoherently Partitioned Pupil masks and show that they significantly extend the DoF of single-molecule localization microscopes. In particular we propose different designs including a simple and easy-to-realize two-ring binary mask to extend the DoF. Moreover, we demonstrate that a simple maximum likelihood-based localization algorithm can reach the localization accuracy predicted by the CRB. The framework developed in this paper is based on an explicit and general information theoretic criterion, and can thus be used as an engineering tool to optimize and compare any type of DoF-enhancing phase mask in high resolution microscopy on a quantitative basis.
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Numerical refocusing in any plane is one powerful feature granted by measuring both the amplitude and the phase of a coherent light beam. Here, we introduce a method based on the first Rytov approximation of scalar electromagnetic fields that (i) allows numerical propagation without requiring phase unwrapping after propagation and (ii) limits the effect of artificial phase singularities that appear upon numerical defocusing when the measurement noise is mixing with the signal. We demonstrate the feasibility of this method with both scalar electromagnetic field simulations and real acquisitions of microscopic biological samples imaged at high numerical aperture.
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We describe a new technique based on the use of a high-resolution quadri-wave lateral shearing interferometer to perform quantitative linear retardance and birefringence measurements on biological samples. The system combines quantitative phase images with varying polarization excitation to create retardance images. This technique is compatible with living samples and gives information about the local retardance and structure of their anisotropic components. We applied our approach to collagen fibers leading to a birefringence value of (3.4 ± 0.3) · 10(-3) and to living cells, showing that cytoskeleton can be imaged label-free.
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We present a full-field technique that allows label-free cytoskeletal network imaging inside living cells. This noninvasive technique allows monitoring of the cytoskeleton dynamics as well as interactions between the latter and organelles on any timescale. It is based on high-resolution quantitative phase imaging (modified Quadriwave lateral shearing interferometry) and can be directly implemented using any optical microscope without modification. We demonstrate the capability of our setup on fixed and living Chinese hamster ovary cells, showing the cytoskeleton dynamics in lamellipodia during protrusion and mitochondria displacement along the cytoskeletal network. In addition, using the quantitative function of the technique, along with simulation tools, we determined the refractive index of a single tubulin microtubule to be ntubu=2.36±0.6 at λ=527 nm.
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Citoesqueleto/metabolismo , Imageamento Tridimensional , Mamíferos/metabolismo , Coloração e Rotulagem , Animais , Células CHO , Cricetinae , Cricetulus , Interferometria , Microscopia de Contraste de Fase , Fenômenos ÓpticosRESUMO
We describe the use of spatially incoherent illumination to make quantitative phase imaging of a semi-transparent sample, even out of the paraxial approximation. The image volume electromagnetic field is collected by scanning the image planes with a quadriwave lateral shearing interferometer, while the sample is spatially incoherently illuminated. In comparison to coherent quantitative phase measurements, incoherent illumination enriches the 3D collected spatial frequencies leading to 3D resolution increase (up to a factor 2). The image contrast loss introduced by the incoherent illumination is simulated and used to compensate the measurements. This restores the quantitative value of phase and intensity. Experimental contrast loss compensation and 3D resolution increase is presented using polystyrene and TiO(2) micro-beads. Our approach will be useful to make diffraction tomography reconstruction with a simplified setup.
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Quantitative phase imaging enables precise and label-free characterizations of individual nano-objects within a large volume, without a priori knowledge of the sample or imaging system. While emerging common path implementations are simple enough to promise a broad dissemination, their phase sensitivity still falls short of precisely estimating the mass or polarizability of vesicles, viruses, or nanoparticles in single-shot acquisitions. In this paper, we revisit the Zernike filtering concept, originally crafted for intensity-only detectors, with the aim of adapting it to wavefront imaging. We demonstrate, through numerical simulation and experiments based on high-resolution wavefront sensing, that a simple Fourier-plane add-on can significantly enhance phase sensitivity for subdiffraction objectsâachieving over an order of magnitude increase (×12)âwhile allowing the quantitative retrieval of both intensity and phase. This advancement allows for more precise nano-object detection and metrology.
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Ultrafast laser processing can induce surface nanostructurating (SNS) in most materials with dimensions close to the irradiation laser wavelength. In-situ SNS characterization could be key for laser parameter's fine-tuning, essential for the generation of complex and/or hybrid nanostructures. Laser Induced Periodic Surface Structures (LIPSS) created in the ultra-violet (UV) range generate the most fascinating effects. They are however highly challenging to characterize in a non-destructive manner since their dimensions can be as small as 100 nm. Conventional optical imaging methods are indeed limited by diffraction to a resolution of [Formula: see text] nm. Although optical super-resolution techniques can go beyond the diffraction limit, which in theory allows the visualization of LIPSS, most super-resolution methods require the presence of small probes (such as fluorophores) which modifies the sample and is usually incompatible with a direct surface inspection. In this paper, we demonstrate that a modified label-free Confocal Reflectance Microscope (CRM) in a photon reassignment regime (also called re-scan microscopy) can detect sub-diffraction limit LIPSS. SNS generated on a titanium sample irradiated with a [Formula: see text] nm femtosecond UV-laser were characterized with nanostructuring period ranging from 105 to 172 nm. Our label-free, non-destructive optical surface inspection was done at 180 [Formula: see text]m[Formula: see text]/s, and the results are compared with commercial SEM showing the metrological efficiency of our approach.
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A generalized product-of-convolution model for simulation of quantitative phase microscopy of thick heterogeneous specimen under tilted plane-wave illumination is presented. Actual simulations are checked against a much more time-consuming commercial finite-difference time-domain method. Then modeled data are compared with experimental measurements that were made with a quadriwave lateral shearing interferometer.
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A wavefront sensor is used as a direct observation tool to image the Gouy phase shift in photonic nanojets created by micrometer-sized dielectric spheres. The amplitude and phase distributions of light are found in good agreement with a rigorous electromagnetic computation. Interestingly the observed phase shift when travelling through the photonic jet is a combination of the awaited π Gouy shift and a phase shift induced by the bead refraction. Such direct spatial phase shift observation using wavefront sensors would find applications in microscopy, diffractive optics, optical trapping, and point spread function engineering.
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We propose and implement a wide-field microscopy method to retrieve the real and imaginary part of a field emitted by coherent and resonant molecular scatterers. The technique is based on wave-front sensing and does not require the use of any reference beam. We exemplify its ability in wide-field coherent anti-Stokes Raman scattering imaging and retrieve the complex anti-Stokes field while spectrally scanning a molecular vibrational resonance. This approach gives access to the background-free Raman spectrum of the targeted molecular bond.
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Wavefront sensors are usually based on measuring the wavefront derivatives. The most commonly used approach to quantitatively reconstruct the wavefront uses discrete Fourier transform, which leads to artifacts when phase objects are located at the image borders. We propose here a simple approach to avoid these artifacts based on the duplication and antisymmetrization of the derivatives data, in the derivative direction, before integration. This approach completely erases the border effects by creating continuity and differentiability at the edge of the image. We finally compare this corrected approach to the literature on model images and quantitative phase images of biological microscopic samples, and discuss the effects of the artifacts on the particular application of dry mass measurements.
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In this Perspective we propose our current point of view and a suggestive roadmap on the field of high-resolution optical microscopy dedicated to bioimaging. Motivated by biological applications, researchers have indeed devised an impressive amount of strategies to address the diverse constraints of imaging and studying biological matter down to the molecular scale, making this interdisciplinary research field a vibrant forum for creativity. Throughout the discussion, we highlight several striking recent successes in this quest. We also identify some next challenges still ahead to apprehend biological questions in increasingly complex living organisms for integrative studies in a minimally invasive manner.
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Phase imaging with a high-resolution wavefront sensor is considered. This is based on a quadriwave lateral shearing interferometer mounted on a non-modified transmission white-light microscope. The measurement technology is explained both in the scope of wave optics and geometrical optics in order to discuss its implementation on a conventional microscope. In particular we consider the effect of a non spatially coherent source on the phase-image signal-to-noise ratio. Precise measurements of the phase-shift introduced by microscopic beads or giant unilamellar vesicles validate the principle and show the accuracy of the methods. Diffraction limited images of living COS-7 cells are then presented, with a particular focus on the membrane and organelle dynamics.
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Interferometria/métodos , Microscopia de Contraste de Fase/métodos , Microscopia/métodos , Óptica e Fotônica , Algoritmos , Animais , Células COS , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Chlorocebus aethiops , Campos Eletromagnéticos , Desenho de Equipamento , Interferometria/instrumentação , Microscopia de Contraste de Fase/instrumentação , Modelos Estatísticos , Organelas/metabolismoRESUMO
The inclusion behavior of natural cyclodextrins (CDs) and polymers based on natural cyclodextrins (CD-polymer), in solution and in solid-state, was studied towards a poorly water-soluble anti-helminthic drug, albendazole (ABZ), chemically methyl[5-(propylthio)-1-H-benzimidazol-2yl]carbamate. Drug-cyclodextrin solid systems were prepared by freeze-drying. Phase solubility study was used to evaluate the interaction in solution, between ABZ/(CDs) and ABZ/CD-polymers. The stability constants of ABZ/natural CDs and ABZ/CD-polymers complexes were calculated by phase solubility method. The apparent solubility of Albendazole was enhanced especially with poly alpha-CD. The formation of inclusion complexes with natural CDs and polymers of cyclodextrin in the solid-state form were confirmed by Fourier Fransform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC) and proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR).
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Cryptosporidiosis, the leading cause of endemic and epidemic diarrhoeal disease worldwide is due to Cryptosporidium parvum, a spore-forming protozoan. Anticryptosporidial pharmacological and/or immunological agents were initially tested in immunodeficient models of cryptosporidiosis, and sinefungin exhibited a significant dose dependent curative and preventive activity. Shedding relapses observed after discontinuation of sinefungin therapy lead to identify the bile ducts as a protected reservoir that may sustain chronic infection. The MIC50 of the sinefungin observed in vitro studies is 14.5 microg/ml. It is a hydrophilic drug lowly absorbed when orally administered and nephrotoxic after IV injection. To avoid this toxic effect, Neal's team prepared sinefungin loaded microspheres and proved that encapsulated sinefungin was 10 times more effective than free sinefungin. This optimistic result led us to study the nanoparticles as drug carrier for sinefungin. In this study, we prepared optimal sinefungin loaded PLGA nanoparticles. Physico-chemical characterization, in vitro drug release and in vivo studies were assessed. Negative surface-charged (-56.1 mV) sinefungin loaded PLGA nanoparticles were prepared with a homogenous size of 200 nm. Optimal formulation led to a drug content of 9.18% w/w (4.59 mg) and a drug entrapment of 15.16%. Dilution technique was used to study the release of sinefungin in vitro. 93.03% of sinefungin were released from dilution 1:5 to 1:20. This burst effect could probably due to the adsorption of the drug on the surface of the nanoparticles. A second step with a lower release was observed from dilution 1:20 to 1:100 which may correspond to the diffusion out of the drug solution from the nanoparticles into the bulk release medium. Investigations in rats showed that only 1.23 mg of sinefungin loaded in PLGA nanoparticles led to a decrease of sinefungin in the urine (0.23 mg vs. 4.27 mg for IV administration of free sinefungin) and to an increase of sinefungin concentration in the bile (6.63 microg/ml vs. 3.89 microg/ml for IV administration of free sinefungin). But the biliary concentration of encapsulated sinefungin (6.63 microg/ml) is still nearly 2 times lower than the MIC50.