RESUMO
Optical imaging through complex media, such as biological tissues or fog, is challenging due to light scattering. In the multiple scattering regime, wavefront shaping provides an effective method to retrieve information; it relies on measuring how the propagation of different optical wavefronts are impacted by scattering. Based on this principle, several wavefront shaping techniques were successfully developed, but most of them are highly invasive and limited to proof-of-principle experiments. Here, we propose to use a neural network approach to non-invasively characterize and control light scattering inside the medium and also to retrieve information of hidden objects buried within it. Unlike most of the recently-proposed approaches, the architecture of our neural network with its layers, connected nodes and activation functions has a true physical meaning as it mimics the propagation of light in our optical system. It is trained with an experimentally-measured input/output dataset built from a series of incident light patterns and corresponding camera snapshots. We apply our physics-based neural network to a fluorescence microscope in epi-configuration and demonstrate its performance through numerical simulations and experiments. This flexible method can include physical priors and we show that it can be applied to other systems as, for example, non-linear or coherent contrast mechanisms.
Assuntos
Dispositivos Ópticos , Física , Microscopia de Fluorescência , Redes Neurais de Computação , Imagem ÓpticaRESUMO
Raman scattering is a chemically selective probing mechanism with diverse applications in industry and clinical settings. Yet, most samples are optically opaque limiting the applicability of Raman probing at depth. Here, we demonstrate chemically selective energy deposition behind a scattering medium by combining prior information on the chemical's spectrum with the measurement of a spectrally resolved Raman speckle as a feedback mechanism for wavefront shaping. We demonstrate unprecedented sixfold signal enhancement in an epi-geometry, realizing targeted energy deposition and focusing on individual Raman active particles.
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Análise Espectral Raman , Fenômenos FísicosRESUMO
The surgical treatments of injured soft tissues lead to further injury due to the use of sutures or the surgical routes, which need to be large enough to insert biomaterials for repair. In contrast, the use of low viscosity photopolymerizable hydrogels that can be inserted with thin needles represents a less traumatic treatment and would therefore reduce the severity of iatrogenic injury. However, the delivery of light to solidify the inserted hydrogel precursor requires a direct access to it, which is mostly invasive. To circumvent this limitation, we investigate the approach of curing the hydrogel located behind biological tissues by sending near-infrared (NIR) light through the latter, as this spectral region has the largest transmittance in biological tissues. Upconverting nanoparticles (UCNPs) are incorporated in the hydrogel precursor to convert NIR transmitted through the tissues into blue light to trigger the photopolymerization. We investigated the photopolymerization process of an adhesive hydrogel placed behind a soft tissue. Bulk polymerization was achieved with local radiation of the adhesive hydrogel through a focused light system. Thus, unlike the common methods for uniform illumination, adhesion formation was achieved with local micrometer-sized radiation of the bulky hydrogel through a gradient photopolymerization phenomenon. Nanoindentation and upright microscope analysis confirmed that the proposed approach for indirect curing of hydrogels below the tissue is a gradient photopolymerization phenomenon. Moreover, we found that the hydrogel mechanical and adhesive properties can be modulated by playing with different parameters of the system such as the NIR light power and the UCNP concentration. The proposed photopolymerization of adhesive hydrogels below the tissue opens the prospect of a minimally invasive surgical treatment of injured soft tissues.
Assuntos
Hidrogéis , Nanopartículas , Adesivos , Materiais Biocompatíveis , PolimerizaçãoRESUMO
Propagation of an ultrashort pulse of light through strongly scattering media generates an intricate spatio-spectral speckle that can be described by means of the multi-spectral transmission matrix (MSTM). In conjunction with a spatial light modulator, the MSTM enables the manipulation of the pulse leaving the medium; in particular focusing it at any desired spatial position and/or time. Here, we demonstrate how to engineer the point-spread-function of the focused beam both spatially and spectrally, from the measured MSTM. It consists of numerically filtering the spatial content at each wavelength of the matrix prior to focusing. We experimentally report on the versatility of the technique through several examples, in particular as an alternative to simultaneous spatial and temporal focusing, with potential applications in multiphoton microscopy.
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Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, a leading cause of cancer-related deaths globally. Initial lesions of PDAC develop within the exocrine pancreas' functional units, with tumor progression driven by interactions between PDAC and stromal cells. Effective therapies require anatomically and functionally relevantin vitrohuman models of the pancreatic cancer microenvironment. We employed tomographic volumetric bioprinting, a novel biofabrication method, to create human fibroblast-laden constructs mimicking the tubuloacinar structures of the exocrine pancreas. Human pancreatic ductal epithelial (HPDE) cells overexpressing the KRAS oncogene (HPDE-KRAS) were seeded in the multiacinar cavity to replicate pathological tissue. HPDE cell growth and organization within the structure were assessed, demonstrating the formation of a thin epithelium covering the acini inner surfaces. Immunofluorescence assays showed significantly higher alpha smooth muscle actin (α-SMA) vs. F-actin expression in fibroblasts co-cultured with cancerous versus wild-type HPDE cells. Additionally,α-SMA expression increased over time and was higher in fibroblasts closer to HPDE cells. Elevated interleukin (IL)-6 levels were quantified in supernatants from co-cultures of stromal and HPDE-KRAS cells. These findings align with inflamed tumor-associated myofibroblast behavior, serving as relevant biomarkers to monitor early disease progression and target drug efficacy. To our knowledge, this is the first demonstration of a 3D bioprinted model of exocrine pancreas that recapitulates its true 3-dimensional microanatomy and shows tumor triggered inflammation.
Assuntos
Bioimpressão , Fibroblastos , Pâncreas Exócrino , Humanos , Pâncreas Exócrino/metabolismo , Fibroblastos/metabolismo , Fibroblastos/citologia , Impressão Tridimensional , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Tomografia , Actinas/metabolismo , Interleucina-6/metabolismo , Engenharia Tecidual , Técnicas de Cocultura , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
3D printing has revolutionized the manufacturing of volumetric components and structures in many areas. Several fully volumetric light-based techniques have been recently developed thanks to the advent of photocurable resins, promising to reach unprecedented short print time (down to a few tens of seconds) while keeping a good resolution (around 100 µm). However, these new approaches only work with homogeneous and relatively transparent resins so that the light patterns used for photo-polymerization are not scrambled along their propagation. Herein, a method that takes into account light scattering in the resin prior to computing projection patterns is proposed. Using a tomographic volumetric printer, it is experimentally demonstrated that implementation of this correction is critical when printing objects whose size exceeds the scattering mean free path. To show the broad applicability of the technique, functional objects of high print fidelity are fabricated in hard organic scattering acrylates and soft cell-laden hydrogels (at 4 million cells mL-1 ). This opens up promising perspectives in printing inside turbid materials with particular interesting applications for bioprinting cell-laden constructs.
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Bioimpressão , Bioimpressão/métodos , Hidrogéis/química , Impressão TridimensionalRESUMO
Non-invasive optical imaging techniques are essential diagnostic tools in many fields. Although various recent methods have been proposed to utilize and control light in multiple scattering media, non-invasive optical imaging through and inside scattering layers across a large field of view remains elusive due to the physical limits set by the optical memory effect, especially without wavefront shaping techniques. Here, we demonstrate an approach that enables non-invasive fluorescence imaging behind scattering layers with field-of-views extending well beyond the optical memory effect. The method consists in demixing the speckle patterns emitted by a fluorescent object under variable unknown random illumination, using matrix factorization and a novel fingerprint-based reconstruction. Experimental validation shows the efficiency and robustness of the method with various fluorescent samples, covering a field of view up to three times the optical memory effect range. Our non-invasive imaging technique is simple, neither requires a spatial light modulator nor a guide star, and can be generalized to a wide range of incoherent contrast mechanisms and illumination schemes.
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Iluminação , Imagem Óptica , Imagem Óptica/métodosRESUMO
In biological microscopy, light scattering represents the main limitation to image at depth. Recently, a set of wavefront shaping techniques has been developed in order to manipulate coherent light in strongly disordered materials. The Transmission Matrix approach has shown its capability to inverse the effect of scattering and efficiently focus light. In practice, the matrix is usually measured using an invasive detector or low-resolution acoustic guide stars. Here, we introduce a non-invasive and all-optical strategy based on linear fluorescence to reconstruct the transmission matrices, to and from a fluorescent object placed inside a scattering medium. It consists in demixing the incoherent patterns emitted by the object using low-rank factorizations and phase retrieval algorithms. We experimentally demonstrate the efficiency of this method through robust and selective focusing. Additionally, from the same measurements, it is possible to exploit memory effect correlations to image and reconstruct extended objects. This approach opens up a new route towards imaging in scattering media with linear or non-linear contrast mechanisms.
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Microscopia de Fluorescência/instrumentação , Algoritmos , Fluorescência , Corantes Fluorescentes/química , Pólen/química , Sementes/químicaRESUMO
Improving the imaging speed of multiphoton microscopy is an active research field. Among recent strategies, light-sheet illumination holds distinctive advantages for achieving fast imaging in vivo. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to find the best balance between signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast live tissue imaging.