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Under spatially incoherent illumination, time-domain full-field optical coherence tomography (FFOCT) offers the possibility to achieve in vivo retinal imaging at cellular resolution over a wide field of view. Such performance is possible, albeit there is the presence of ocular aberrations even without the use of classical adaptive optics. While the effect of aberrations in FFOCT has been debated these past years, mostly on low-order and static aberrations, we present, for the first time to our knowledge, a method enabling a quantitative study of the effect of statistically representative static and dynamic ocular aberrations on FFOCT image metrics, such as SNR, resolution, and image similarity. While we show that ocular aberrations can decrease FFOCT SNR and resolution by up to 14 dB and fivefold, we take advantage of such quantification to discuss different possible compromises between performance gain and adaptive optics complexity and speed, to optimize both sensor-based and sensorless FFOCT high-resolution retinal imaging.
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Retina , Tomografia de Coerência Óptica , Tomografia de Coerência Óptica/métodos , Retina/diagnóstico por imagem , Humanos , Razão Sinal-RuídoRESUMO
We demonstrate the feasibility of a multimodal adaptive optics flood-illumination ophthalmoscope, able to provide both bright-field and dark-field images (such as phase contrast). The multimodality was made possible by integrating a digital micromirror device (DMD) at the illumination path to project a sequence of complementary high-resolution patterns into the retina. Through a versatile post-processing method that digitally selects backscattered or multiply scattered photons, we were able: (1) to achieve up to four-fold contrast increase of bright-field images when imaging the photoreceptor mosaic and nerve fibers; and (2) to visualize translucent retinal features such as capillaries, red blood cells, vessel walls, ganglion cells, and photoreceptor inner segments through phase contrast.
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Iluminação , Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Cones/fisiologia , Inundações , Tomografia de Coerência Óptica/métodos , Retina/diagnóstico por imagem , OftalmoscópiosRESUMO
Effective and accurate in vivo diagnosis of retinal pathologies requires high performance imaging devices, combining a large field of view and the ability to discriminate the ballistic signal from the diffuse background in order to provide a highly contrasted image of the retinal structures. Here, we have implemented the partial-field illumination ophthalmoscope, a patterned illumination modality, integrated to a high pixel rate adaptive optics full-field microscope. This non-invasive technique enables us to mitigate the low signal-to-noise ratio, intrinsic of full-field ophthalmoscopes, by partially illuminating the retina with complementary patterns to reconstruct a wide-field image. This new, to the best of our knowledge, modality provides an image contrast spanning from the full-field to the confocal contrast, depending on the pattern size. As a result, it offers various trade-offs in terms of contrast and acquisition speed, guiding the users towards the most efficient system for a particular clinical application.
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Sensibilidades de Contraste/fisiologia , Iluminação , Oftalmoscópios , Fotografação/instrumentação , Retina/diagnóstico por imagem , Desenho de Equipamento , Humanos , Óptica e Fotônica , Razão Sinal-RuídoRESUMO
Label-free live optical imaging of dynamic cellular and subcellular features has been made possible in recent years thanks to the advances made in optical imaging techniques, including dynamic optical coherence tomography (D-OCT) methods. These techniques analyze the temporal fluctuations of an optical signal associated with the active movements of intracellular organelles to obtain an ensemble metric recapitulating the motility and metabolic state of cells. They hence enable visualization of cells within compact, static environments and evaluate their physiology. These emerging microscopies show promise, in particular for the three-dimensional evaluation of live tissue samples such as freshly excised biopsies and 3D cell cultures. In this review, we compare the various techniques used for dynamic OCT. We give an overview of the range of applications currently being explored and discuss the future outlook and opportunities for the field.
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This work compares two tomographic imaging technologies, time-domain full-field optical coherence tomography (FFOCT) working in reflection and optical transmission tomography (OTT), using a new optical setup that combines both. We show that, due to forward-scattering properties, the axial sectioning and contrast in OTT can be optimized by tuning illumination. The influence of sample scattering and thickness are discussed. We illustrate the comparison of the two methods in static (morphology) and dynamic (metabolic contrast) regimes using cell cultures, tissues and entire organisms emphasizing the advantages of both approaches.
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Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as an invaluable live label-free and non-invasive imaging modality able to image subcellular biological structures and their metabolic activity within complex 3D samples. However, D-FFOCT suffers from fringe artefacts when imaging near reflective surfaces and is highly sensitive to vibrations. Here, we present interface Self-Referenced (iSR) D-FFOCT, an alternative configuration to D-FFOCT that takes advantage of the presence of the sample coverslip in between the sample and the objective by using it as a defocused reference arm, thus avoiding the aforementioned artefacts. We demonstrate the ability of iSR D-FFOCT to image 2D fibroblast cell cultures, which are among the flattest mammalian cells.
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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.
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Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening.
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Células-Tronco Pluripotentes Induzidas , Tomografia de Coerência Óptica , Humanos , Tomografia de Coerência Óptica/métodos , Retina , Técnicas de Cultura de Células , OrganoidesRESUMO
Purpose: The adoption of emerging imaging technologies in the medical community is often hampered when they provide a new unfamiliar contrast that requires experience to be interpreted. Dynamic full-field optical coherence tomography (D-FF-OCT) microscopy is such an emerging technique. It provides fast, high-resolution images of excised tissues with a contrast comparable to H&E histology but without any tissue preparation and alteration. Approach: We designed and compared two machine learning approaches to support interpretation of D-FF-OCT images of breast surgical specimens and thus provide tools to facilitate medical adoption. We conducted a pilot study on 51 breast lumpectomy and mastectomy surgical specimens and more than 1000 individual 1.3×1.3 mm2 images and compared with standard H&E histology diagnosis. Results: Using our automatic diagnosis algorithms, we obtained an accuracy above 88% at the image level (1.3×1.3 mm2) and above 96% at the specimen level (above cm2). Conclusions: Altogether, these results demonstrate the high potential of D-FF-OCT coupled to machine learning to provide a rapid, automatic, and accurate histopathology diagnosis with minimal sample alteration.
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The mesencephalic locomotor region (MLR) is a brain stem area whose stimulation triggers graded forward locomotion. How MLR neurons recruit downstream vsx2+ (V2a) reticulospinal neurons (RSNs) is poorly understood. Here, to overcome this challenge, we uncovered the locus of MLR in transparent larval zebrafish and show that the MLR locus is distinct from the nucleus of the medial longitudinal fasciculus. MLR stimulations reliably elicit forward locomotion of controlled duration and frequency. MLR neurons recruit V2a RSNs via projections onto somata in pontine and retropontine areas, and onto dendrites in the medulla. High-speed volumetric imaging of neuronal activity reveals that strongly MLR-coupled RSNs are active for steering or forward swimming, whereas weakly MLR-coupled medullary RSNs encode the duration and frequency of the forward component. Our study demonstrates how MLR neurons recruit specific V2a RSNs to control the kinematics of forward locomotion and suggests conservation of the motor functions of V2a RSNs across vertebrates.
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Mesencéfalo , Peixe-Zebra , Animais , Larva , Mesencéfalo/fisiologia , Locomoção/fisiologia , Neurônios/fisiologia , Medula Espinal/fisiologia , Estimulação ElétricaRESUMO
Introduction: The diagnosis of cutaneous manifestations of deep mycoses relies on both histopathological and direct examinations. Yet, the current diagnostic criteria cannot prevent missed cases, including invasive aspergillosis, which requires the development of a novel diagnostic approach and imaging tools. We recently introduced the use of dynamic full-field optical coherence tomography (D-FF-OCT) in fungal diagnostics with a definition approaching that of conventional microscopy and the ability to return metabolic information regarding different fungal species. The present work focuses on subcellular dynamics and live-cell imaging of Aspergillus fumigatus with D-FF-OCT to follow the fungal growth stages. Methods: The A. fumigatus ATCC 204305 quality-control strain was used for all imaging experiments, following incubation times varying between 24 and 72 h at 30°C in a humidified chamber on Sabouraud dextrose agar. Fungal growth was subsequently monitored with D-FF-OCT for up to 5 h at room temperature and following the pharmacological stress of either voriconazole, amphotericin B, or caspofungin gradient concentration. Results: D-FF-OCT images allow not only the visualization of intracellular trafficking of vacuoles but also an evolving dynamic segmentation of conidiophores depending on the chronological development and aging of the hyphae or the effect of antifungal treatment. The same applies to conidial heads, with the most intense D-FF-OCT signal coming from vesicles, revealing a changing dynamic within a few hours only, as well as complete extinction following subsequent drying of the Sabouraud dextrose agar. Discussion: These results provide additional data on the ability of D-FF-OCT to monitor some of the main life cycle processes, dynamics, and intracellular trafficking of vacuoles in A. fumigatus, with or without the effect of pharmacological stress. Such complementary metabolic information could help both clinicians and microbiologists in either mechanistic studies toward experimental mycology or the development of a potential D-FF-OCT-guided diagnosis of superficial fungal infections.
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Aspergillus fumigatus , Tomografia de Coerência Óptica , Ágar/farmacologia , Antifúngicos/farmacologia , GlucoseRESUMO
There is an increasing need for label free methods that could reveal intracellular structures and dynamics. In this context, we develop a new optical tomography method working in transmission - full-field optical transmission tomography (FF-OTT). The method can measure the forward scattering signals and reveals the time-dependent metabolic signals in living cells. FF-OTT is a common path interferometer taking advantage of the Gouy phase shift - a π phase shift that the light wave experiences around the focus. By modulating the position of the focus one can alter the phase of the scattered light. Demodulation of images with different phases rejects the background and enhances the light from the depth-of-field, thus producing an optical section. We test FF-OTT by imaging single-cell diatoms and ex vivo biological samples. In fresh samples, we show that the intracellular motions create visible intensity fluctuations in FF-OTT so that the method is able to reveal a metabolic dynamic contrast. FF-OTT was found to be an efficient label free technique that can be readily implemented thanks to a robust common-path speckle-free interferometer design using an incoherent light source.
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Circulation of cerebrospinal fluid (CSF) plays an important role during development. In zebrafish embryo, the flow of CSF has been found to be bidirectional in the central canal of the spinal cord. In order to compare conditions and genetic mutants across each other, we recently automated the quantification of the velocity profile of exogenous fluorescent particles in the CSF. We demonstrated that the beating of motile and tilted cilia localized on the ventral side of the central canal was sufficient to generate locally such bidirectionality. Our approach can easily be extended to characterize CSF flow in various genetic mutants. We provide here a detailed protocol and a user interface program to quantify CSF dynamics. In order to interpret potential changes in CSF flow profiles, we provide additional tools to measure the central canal diameter, characterize cilia dynamics and compare experimental data with our theoretical model in order to estimate the impact of cilia in generating a volume force in the central canal. Our approach can also be of use for measuring particle velocity in vivo and modeling flow in diverse biological solutions.
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Allying high-resolution with a large field-of-view (FOV) is of great importance in the fields of biology and medicine, but it is particularly challenging when imaging non-flat living samples such as the human retina. Indeed, high-resolution is normally achieved with adaptive optics (AO) and scanning methods, which considerably reduce the useful FOV and increase the system complexity. An alternative technique is time-domain full-field optical coherence tomography (FF-OCT), which has already shown its potential for in-vivo high-resolution retinal imaging. Here, we introduce coherence gate shaping for FF-OCT, to optically shape the coherence gate geometry to match the sample curvature, thus achieving a larger FOV than previously possible. Using this instrument, we obtained high-resolution images of living human photoreceptors close to the foveal center without AO and with a 1 mm × 1 mm FOV in a single shot. This novel advance enables the extraction of photoreceptor-based biomarkers with ease and spatiotemporal monitoring of individual photoreceptors. We compare our findings with AO-assisted ophthalmoscopes, highlighting the potential of FF-OCT, as a compact system, to become a routine clinical imaging technique.
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Circulation of the cerebrospinal fluid (CSF) contributes to body axis formation and brain development. Here, we investigated the unexplained origins of the CSF flow bidirectionality in the central canal of the spinal cord of 30 hpf zebrafish embryos and its impact on development. Experiments combined with modeling and simulations demonstrate that the CSF flow is generated locally by caudally-polarized motile cilia along the ventral wall of the central canal. The closed geometry of the canal imposes the average flow rate to be null, explaining the reported bidirectionality. We also demonstrate that at this early stage, motile cilia ensure the proper formation of the central canal. Furthermore, we demonstrate that the bidirectional flow accelerates the transport of particles in the CSF via a coupled convective-diffusive transport process. Our study demonstrates that cilia activity combined with muscle contractions sustain the long-range transport of extracellular lipidic particles, enabling embryonic growth.
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Líquido Cefalorraquidiano/fisiologia , Reologia , Medula Espinal/fisiologia , Animais , Animais Geneticamente Modificados , Transporte Biológico , Ventrículos Cerebrais/fisiologia , Cílios/fisiologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Proteínas de Fluorescência Verde/metabolismo , Contração Muscular/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologiaRESUMO
Recent evidence indicates active roles for the cerebrospinal fluid (CSF) on body axis development and morphogenesis of the spine, implying CSF-contacting neurons (CSF-cNs) in the spinal cord. CSF-cNs project a ciliated apical extension into the central canal that is enriched in the channel PKD2L1 and enables the detection of spinal curvature in a directional manner. Dorsolateral CSF-cNs ipsilaterally respond to lateral bending although ventral CSF-cNs respond to longitudinal bending. Historically, the implication of the Reissner fiber (RF), a long extracellular thread in the CSF, to CSF-cN sensory functions has remained a subject of debate. Here, we reveal, using electron microscopy in zebrafish larvae, that the RF is in close vicinity with cilia and microvilli of ventral and dorsolateral CSF-cNs. We investigate in vivo the role of cilia and the RF in the mechanosensory functions of CSF-cNs by combining calcium imaging with patch-clamp recordings. We show that disruption of cilia motility affects CSF-cN sensory responses to passive and active curvature of the spinal cord without affecting the Pkd2l1 channel activity. Because ciliary defects alter the formation of the RF, we investigated whether the RF contributes to CSF-cN mechanosensitivity in vivo. Using a hypomorphic mutation in the scospondin gene that forbids the aggregation of SCO-spondin into a fiber, we demonstrate in vivo that the RF per se is critical for CSF-cN mechanosensory function. Our study uncovers that neurons contacting the cerebrospinal fluid functionally interact with the RF to detect spinal curvature in the vertebrate spinal cord.
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Líquido Cefalorraquidiano/fisiologia , Morfogênese , Células Receptoras Sensoriais/fisiologia , Medula Espinal/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Animais , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Medula Espinal/ultraestruturaRESUMO
We describe recent technological progress in multimodal en face full-field optical coherence tomography that has allowed detection of slow and fast dynamic processes in the eye. We show that by combining static, dynamic and fluorescence contrasts we can achieve label-free high-resolution imaging of the retina and anterior eye with temporal resolution from milliseconds to several hours, allowing us to probe biological activity at subcellular scales inside 3D bulk tissue. Our setups combine high lateral resolution over a large field of view with acquisition at several hundreds of frames per second which make it a promising tool for clinical applications and biomedical studies. Its contactless and non-destructive nature is shown to be effective for both following in vitro sample evolution over long periods of time and for imaging of the human eye in vivo.
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Organ development depends on the integration of coordinated long-range communication between cells. The cerebrospinal fluid composition and flow properties regulate several aspects of central nervous system development, including progenitor proliferation, neurogenesis, and migration [1-3]. One understudied component of the cerebrospinal fluid, described over a century ago in vertebrates, is the Reissner fiber. This extracellular thread forming early in development results from the assembly of the SCO-spondin protein in the third and fourth brain ventricles and central canal of the spinal cord [4]. Up to now, the function of the Reissner fiber has remained elusive, partly due to the lack of genetic invalidation models [4]. Here, by mutating the scospondin gene, we demonstrate that the Reissner fiber is critical for the morphogenesis of a straight posterior body axis. In zebrafish mutants where the Reissner fiber is lost, ciliogenesis and cerebrospinal fluid flow are intact but body axis morphogenesis is impaired. Our results also explain the frequently observed phenotype that mutant embryos with defective cilia exhibit defects in body axis curvature. Here, we reveal that these mutants systematically fail to assemble the Reissner fiber. We show that cilia promote the formation of the Reissner fiber and that the fiber is necessary for proper body axis morphogenesis. Our study sets the stage for future investigations of the mechanisms linking the Reissner fiber to the control of body axis curvature during vertebrate development.
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Padronização Corporal/fisiologia , Líquido Cefalorraquidiano/metabolismo , Cílios/fisiologia , Peixe-Zebra/embriologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Mutação , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298tm304 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSF-cNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine.