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Significance: The reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption. Aim: To prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed. Approach: A deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system. Results: The system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components. Conclusions: An easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.
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Lentes , Dispositivos Ópticos , Animales , Microscopía/métodos , Pez Cebra , Óptica y Fotónica , ÓxidosRESUMEN
The recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock.
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Arabidopsis , Microscopía Fluorescente , Colorantes , Recolección de Datos , Respuesta al Choque TérmicoRESUMEN
Heterogeneity investigation at the single-cell level reveals morphological and phenotypic characteristics in cell populations. In clinical research, heterogeneity has important implications in the correct detection and interpretation of prognostic markers and in the analysis of patient-derived material. Among single-cell analysis, imaging flow cytometry allows combining information retrieved by single cell images with the throughput of fluidic platforms. Nevertheless, these techniques might fail in a comprehensive heterogeneity evaluation because of limited image resolution and bidimensional analysis. Light sheet fluorescence microscopy opened new ways to study in 3D the complexity of cellular functionality in samples ranging from single-cells to micro-tissues, with remarkably fast acquisition and low photo-toxicity. In addition, structured illumination microscopy has been applied to single-cell studies enhancing the resolution of imaging beyond the conventional diffraction limit. The combination of these techniques in a microfluidic environment, which permits automatic sample delivery and translation, would allow exhaustive investigation of cellular heterogeneity with high throughput image acquisition at high resolution. Here we propose an integrated optofluidic platform capable of performing structured light sheet imaging flow cytometry (SLS-IFC). The system encompasses a multicolor directional coupler equipped with a thermo-optic phase shifter, cylindrical lenses and a microfluidic network to generate and shift a patterned light sheet within a microchannel. The absence of moving parts allows a stable alignment and an automated fluorescence signal acquisition during the sample flow. The platform enables 3D imaging of an entire cell in about 1 s with a resolution enhancement capable of revealing sub-cellular features and sub-diffraction limit details.
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Imagenología Tridimensional , Microfluídica , Humanos , Microscopía Fluorescente/métodos , Citometría de Flujo/métodos , Imagenología Tridimensional/métodosRESUMEN
Microfluidics have transformed diagnosis and screening in regenerative medicine. Recently, they are showing much promise in biofabrication. However, their adoption is inhibited by costly and drawn-out lithographic processes thus limiting progress. Here, multi-material fibers with complex core-shell geometries with sizes matching those of human arteries and arterioles are fabricated employing versatile microfluidic devices produced using an agile and inexpensive manufacturing pipeline. The pipeline consists of material extrusion additive manufacturing with an innovative continuously varied extrusion (CONVEX) approach to produce microfluidics with complex seamless geometries including, novel variable-width zigzag (V-zigzag) mixers with channel widths ranging from 100-400 µm and hydrodynamic flow-focusing components. The microfluidic systems facilitated rapid mixing of fluids by decelerating the fluids at specific zones to allow for increased diffusion across the interfaces. Better mixing even at high flow rates (100-1000 µL min-1 ) whilst avoiding turbulence led to high cell cytocompatibility (>86%) even when 100 µm nozzles are used. The presented 3D-printed microfluidic system is versatile, simple and efficient, offering a great potential to significantly advance the microfluidic platform in regenerative medicine.
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Dispositivos Laboratorio en un Chip , Microfluídica , Humanos , Medicina Regenerativa , Impresión Tridimensional , HidrodinámicaRESUMEN
Structured Illumination Microscopy (SIM) is a key technology for high resolution and super-resolution imaging of biological cells and molecules. The spread of portable and easy-to-align SIM systems requires the development of novel methods to generate a light pattern and to shift it across the field of view of the microscope. Here we show a miniaturized chip that incorporates optical waveguides, splitters, and phase shifters, to generate a 2D structured illumination pattern suitable for SIM microscopy. The chip creates three point-sources, coherent and controlled in phase, without the need for further alignment. Placed in the pupil of a microscope's objective, the three sources generate a hexagonal illumination pattern on the sample, which is spatially translated thanks to thermal phase shifters. We validate and use the chip, upgrading a commercial inverted fluorescence microscope to a SIM setup and we image biological sample slides, extending the resolution of the microscope.
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Iluminación , Dispositivos Ópticos , Microscopía Fluorescente/métodosRESUMEN
Single-cell imaging and sorting are critical technologies in biology and clinical applications. The power of these technologies is increased when combined with microfluidics, fluorescence markers, and machine learning. However, this quest faces several challenges. One of these is the effect of the sample flow velocity on the classification performances. Indeed, cell flow speed affects the quality of image acquisition by increasing motion blur and decreasing the number of acquired frames per sample. We investigate how these visual distortions impact the final classification task in a real-world use-case of cancer cell screening, using a microfluidic platform in combination with light sheet fluorescence microscopy. We demonstrate, by analyzing both simulated and experimental data, that it is possible to achieve high flow speed and high accuracy in single-cell classification. We prove that it is possible to overcome the 3D slice variability of the acquired 3D volumes, by relying on their 2D sum z-projection transformation, to reach an efficient real time classification with an accuracy of 99.4% using a convolutional neural network with transfer learning from simulated data. Beyond this specific use-case, we provide a web platform to generate a synthetic dataset and to investigate the effect of flow speed on cell classification for any biological samples and a large variety of fluorescence microscopes (https://www.creatis.insa-lyon.fr/site7/en/MicroVIP).
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Algoritmos , Microfluídica , Aprendizaje Automático , Microscopía Fluorescente , Redes Neurales de la ComputaciónRESUMEN
Lung carcinoids are neuroendocrine tumors that comprise well-differentiated typical (TCs) and atypical carcinoids (ACs). Preclinical models are indispensable for cancer drug screening since current therapies for advanced carcinoids are not curative. We aimed to develop a novel in vivo model of lung carcinoids based on the xenograft of lung TC (NCI-H835, UMC-11, and NCI-H727) and AC (NCI-H720) cell lines and patient-derived cell cultures in Tg(fli1a:EGFP)y1 zebrafish embryos. We exploited this platform to test the anti-tumor activity of sulfatinib. The tumorigenic potential of TC and AC implanted cells was evaluated by the quantification of tumor-induced angiogenesis and tumor cell migration as early as 24 h post-injection (hpi). The characterization of tumor-induced angiogenesis was performed in vivo and in real time, coupling the tumor xenograft with selective plane illumination microscopy on implanted zebrafish embryos. TC-implanted cells displayed a higher pro-angiogenic potential compared to AC cells, which inversely showed a relevant migratory behavior within 48 hpi. Sulfatinib inhibited tumor-induced angiogenesis, without affecting tumor cell spread in both TC and AC implanted embryos. In conclusion, zebrafish embryos implanted with TC and AC cells faithfully recapitulate the tumor behavior of human lung carcinoids and appear to be a promising platform for drug screening.
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Tumor Carcinoide , Carcinoma Neuroendocrino , Neoplasias Pulmonares , Animales , Tumor Carcinoide/tratamiento farmacológico , Carcinoma Neuroendocrino/patología , Xenoinjertos , Humanos , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Pez CebraRESUMEN
Physical methods to control pest arthropods are increasing in importance, but detailed knowledge of the effects of some of these methods on the target organisms is lacking. The aim of this study was to use light sheet fluorescence microscopy (LSFM) in anatomical studies of blood-sucking arthropods in vivo to assess the suitability of this method to investigate the morphological structures of arthropods and changes in these structures over time, using the human louse Pediculus humanus (Phthiraptera: Pediculidae) as sample organism. Plasma treatment was used as an example of a procedure employed to control arthropods. The lice were prepared using an artificial membrane feeding method involving the ingestion of human blood alone and human blood with an added fluorescent dye in vitro. It was shown that such staining leads to a notable enhancement of the imaging contrast with respect to unstained whole lice and internal organs that can normally not be viewed by transmission microscopy but which become visible by this approach. Some lice were subjected to plasma treatment to inflict damage to the organisms, which were then compared to untreated lice. Using LSFM, a change in morphology due to plasma treatment was observed.These results demonstrate that fluorescence staining coupled with LSFM represents a powerful and straightforward method enabling the investigation of the morphology-including anatomy-of blood-sucking lice and other arthropods.
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Artrópodos , Infestaciones por Piojos , Pediculus , Animales , Colorantes , Ingestión de Alimentos , Humanos , Membranas Artificiales , Microscopía FluorescenteRESUMEN
Combining the information coming from multiview acquisitions is a problem of great interest in light-sheet microscopy. Aligning the views and increasing the resolution of their fusion can be challenging, especially if the setup is not fully calibrated. Here, we tackle these issues by proposing a new reconstruction method based on autocorrelation inversion that avoids alignment procedures. On top of this, we add a blind deconvolution step to improve the resolution of the final reconstruction. Our method permits us to achieve inherently aligned, highly resolved reconstructions while, at the same time, estimating the unknown point-spread function of the system. RESEARCH HIGHLIGHTS: We tackle the problem of multiview light-sheet deconvolution with a blind approach of autocorrelation inversion Our method recovers the object and PSF, requires no alignment and calibration, and enhances the reconstruction of the specimen.
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Aumento de la Imagen , Microscopía , Humanos , Aumento de la Imagen/métodos , Microscopía/métodosRESUMEN
A multimodal imaging study of chabazite is used to show the distribution of and discriminate between different emissive deposits arising as a result of the detemplation process. Confocal imaging, 3D fluorescence lifetime imaging, 3D multispectral fluorescence imaging, and Raman mapping are used to show three different types of emissive behaviours each characterised by different spatial distributions, trends in lifetime, spectral signals, and Raman signatures. A notable difference is seen in the morphology of agglomerated surface deposits and larger subsurface deposits, which experience lifetime augmentation due to spatial confinement. The distribution of organic residue throughout the crystal volume is comparable to XRF mapping that shows Si enrichment on the outer edges and higher Al content through the centre, demonstrating that a fluorescence-based technique can also be used to indirectly comment on the compositional chemistry of the inorganic framework.
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The range of cellular functions the mechanistic target of rapamycin (mTOR) protein performs makes it an attractive drug target for cancer therapy. However, the cellular localisation and mode of action of second generation inhibitors of mTOR is poorly understood despite the level of attention there is in targeting the mTOR protein. We have therefore studied the properties of the pan-mTOR inhibitor AZD2014, an ideal candidate to study because it is naturally fluorescent, characterising its photochemical properties in solution phase (DMSO, PBS and BSA) and within living cells, where it localises within both the nucleus and the cytoplasm but with different excited state lifetimes of 4.8 (+/- 0.5) and 3.9 (+/- 0.4) ns respectively. We measure the uptake of the inhibitor AZD2014 (7 µM) in monolayer HEK293 cells occurring with a half-life of 1 min but observe complex behaviour for 3D spheroids with the core of the spheroid showing a slower uptake and a slow biphasic behaviour at longer times. From a cellular perspective using fluorescence lifetime imaging microscopy AZD2014 was found to interact directly with GFP-tagged mTORC1 proteins including the downstream target, S6K1. We observe light sensitive behaviour of the cells containing AZD2014 which leads to cell death, in both monolayer and spheroids cells, demonstrating the potential of AZD2014 to act as a possible photodynamic drug under both single photon and multiphoton excitation and discuss its use as a photosensitizer. We also briefly characterise another pan-mTOR inhibitor, INK128.
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Antineoplásicos/química , Benzamidas/química , Colorantes Fluorescentes/química , Morfolinas/química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Apoptosis , Benzamidas/farmacología , Benzoxazoles/química , Benzoxazoles/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Proliferación Celular , Cricetulus , Humanos , Cinética , Microscopía de Fluorescencia por Excitación Multifotónica , Morfolinas/farmacología , Imagen Óptica , Fotoquimioterapia , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacologíaRESUMEN
Optical Projection Tomography (OPT) is a powerful three-dimensional imaging technique used for the observation of millimeter-scaled biological samples, compatible with bright-field and fluorescence contrast. OPT is affected by spatially variant artifacts caused by the fact that light diffraction is not taken into account by the straight-light propagation models used for reconstruction. These artifacts hinder high-resolution imaging with OPT. In this work we show that, by using a multiview imaging approach, a 3D reconstruction of the bright-field contrast can be obtained without the diffraction artifacts typical of OPT, drastically reducing the amount of acquired data, compared to previously reported approaches. The method, purely based on bright-field contrast of the unstained sample, provides a comprehensive picture of the sample anatomy, as demonstrated in vivo on Arabidopsis thaliana and zebrafish embryos. Furthermore, this bright-field reconstruction can be implemented on practically any multi-view light-sheet fluorescence microscope without complex hardware modifications or calibrations, complementing the fluorescence information with tissue anatomy.
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In time-domain diffuse optical spectroscopy, the simultaneous acquisition of the time-of-flight distribution (DTOF) of photons traveling in a diffusive medium and of the instrument response function (IRF) is necessary to perform quantitative measurements of optical properties (absorption and reduced scattering coefficients) while taking into account the non-idealities of a real system (e.g. temporal resolution and time delays). The IRF acquisition can be a non-trivial and time-consuming operation that requires directly facing the injection and collection fibers. Since this operation is not always possible, a new IRF measurement scheme is here proposed where the IRF is acquired in reflectance geometry from a corrugate reflective surface. Validation measurements on a set of reference homogenous phantoms have been performed, resulting in an error in the optical properties estimation lower than 10% with respect to the typical IRF configuration. Thus, the proposed method proved to be a reliable approach that after a preliminary calibration can be exploited in a laboratory and clinical set-ups, leading to faster and more accurate measurements and reducing the operator-dependent performance.
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Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for l-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.
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Aminoácidos/metabolismo , Proteínas de Arabidopsis/ultraestructura , Arabidopsis/metabolismo , Dominios Proteicos/genética , Receptores de Glutamato/ultraestructura , Arabidopsis/genética , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión/genética , Calcio/metabolismo , Cristalografía por Rayos X , Citosol/metabolismo , Ligandos , Mutación , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Plantones/metabolismo , Relación Estructura-ActividadRESUMEN
Light sheet fluorescence microscopy has become one of the most widely used techniques for three-dimensional imaging due to its high speed and low phototoxicity. Further improvements in 3D microscopy require limiting the light exposure of the sample and increasing the volumetric acquisition rate. We hereby present an imaging technique that allows volumetric reconstruction of the fluorescent sample using spatial modulation on a selective illumination volume. We demonstrate that this can be implemented using an incoherent LED source, avoiding shadowing artifacts, typical of light sheet microscopy. Furthermore, we show that spatial modulation allows the use of Compressive Sensing, reducing the number of modulation patterns to be acquired. We present results on zebrafish embryos which prove that selective spatial modulation can be used to reconstruct relatively large volumes without any mechanical movement. The technique yields an accurate reconstruction of the sample anatomy even at significant compression ratios, achieving higher volumetric acquisition rate and reducing photodamage biological samples.
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Adipose models have been applied to mechanistic studies of metabolic diseases (such as diabetes) and the subsequent discovery of new therapeutics. However, typical models are either insufficiently complex (2D cell cultures) or expensive and labor intensive (mice/in vivo). To bridge the gap between these models and in order to better inform pre-clinical studies we have developed a drug-responsive 3D model of white adipose tissue (WAT). Here, spheroids (680 ± 60 µm) comprising adipogenic 3T3-L1 cells encapsulated in 3D matrix were fabricated manually on a 96 well scale. Spheroids were highly characterised for lipid morphology, selected metabolite and adipokine secretion, and gene expression; displaying significant upregulation of certain adipogenic-specific genes compared with a 2D model. Furthermore, induction of lipolysis and promotion of lipogenesis in spheroids could be triggered by exposure to 8-br-cAMP and oleic-acid respectively. Metabolic and high content imaging data of spheroids exposed to an adipose-targeting drug, rosiglitazone, resulted in dose-responsive behavior. Thus, our 3D WAT model has potential as a powerful scalable tool for compound screening and for investigating adipose biology.
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Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipoquinas/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Evaluación Preclínica de Medicamentos/instrumentación , Ratones , Rosiglitazona/farmacología , Esferoides Celulares/citología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismoRESUMEN
The first detailed analysis of FLIM applications for Mg cell imaging is presented. We employed the Mg-sensitive fluorescent dye named DCHQ5, a derivative of diaza-18-crown-6 ethers appended with two 8-hydroxyquinoline groups, to perform fluorescence lifetime imaging in control and Mg deprived SaOS-2 live cells, which contain different concentrations of magnesium. We found that the lifetime maps are almost uniform all over the cells and, most relevantly, we showed that the ratio of the amplitude terms is related to the magnesium intracellular concentration.
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Neoplasias Óseas/metabolismo , Magnesio/metabolismo , Imagen Óptica/métodos , Osteosarcoma/metabolismo , Espectrometría de Fluorescencia/métodos , Humanos , Magnesio/análisis , Células Tumorales CultivadasRESUMEN
Calcium imaging in plants requires a high-resolution microscope, able to perform volumetric acquisition in a few seconds, inducing as low photobleaching and phototoxicity as possible to the sample. Light sheet fluorescence microscopy offers these capabilities, with the further chance to mount the sample in vertical position, mimicking the plant's growth and physiological conditions.A protocol for plant preparation and mounting in a light sheet microscope is presented. First, the growth of Arabidopsis thaliana in a sample holder compatible with light sheet microscopy is described. Then, the requirements for sample alignment and image acquisition are detailed. Finally, the image processing steps to analyze calcium oscillations are discussed, with particular emphasis on ratiometric calcium imaging in Arabidopsis root hairs.
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Arabidopsis/metabolismo , Señalización del Calcio , Calcio/metabolismo , Microscopía Fluorescente/métodos , Raíces de Plantas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Luz , Imagen Óptica/métodos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/ultraestructuraRESUMEN
Understanding the dynamic cellular behaviours driving morphogenesis and regeneration is a long-standing challenge in biology. Live imaging, together with genetically encoded reporters, may provide the necessary tool to address this issue, permitting the in vivo monitoring of the spatial and temporal expression dynamics of a gene of interest during a variety of developmental processes. Canonical Wnt/ß-catenin signalling controls a plethora of cellular activities during development, regeneration and adulthood throughout the animal kingdom. Several reporters have been produced in animal models to reveal sites of active Wnt signalling. In order to monitor in vivo Wnt/ß-catenin signalling activity in the freshwater polyp Hydra vulgaris, we generated a ß-cat-eGFP transgenic Hydra, in which eGFP is driven by the Hydra ß-catenin promoter. We characterized the expression dynamics during budding, regeneration and chemical activation of the Wnt/ß-cat signalling pathway using light sheet fluorescence microscopy. Live imaging of the ß-cat-eGFP lines recapitulated the previously reported endogenous expression pattern of ß-catenin and revealed the dynamic appearance of novel sites of Wnt/ß-catenin signalling, that earlier evaded detection by mean of in situ hybridization. By combining the Wnt activity read-out efficiency of the ß-catenin promoter with advanced imaging, we have created a novel model system to monitor in real time the activity of Hydra ß-cat regulatory sequences in vivo, and open the path to reveal ß-catenin modulation in many other physiological contexts.