Advances in fluorescence microscopy have reached a plateau in improving depth-to-resolution ratios for imaging scattering tissues, highlighting an urgent need for innovative techniques. We introduce Wavelens, a groundbreaking opaque lens that combines dynamic wavefront shaping with a passive, angularly anisotropic photonic scattering plate-designed to scatter light predominantly in one direction. This compact, robust optoelectronic device, comparable in size to a conventional microscope lens, delivers superior light sheet formation capabilities. Using Wavelens, we achieved high-resolution LSFM imaging of diverse biological specimens, including 3D tumor spheroids and the intricate neural architecture of Caenorhabditis elegans in vivo. Notably, Wavelens maintains consistent light sheet properties even in dense, scattering-prone samples, overcoming a long-standing limitation of traditional microscopy. This innovative approach has the potential to revolutionize optical imaging, offering unprecedented clarity and resilience, even within dense, scattering-prone biological samples.

We present Wavelens, a compact, microscope objective-like wavefront-shaping optical device for tailored illumination in Light-Sheet Fluorescence Microscopy (LSFM). We characterize its performance and demonstrate superior in vivo imaging of fluorescent specimens compared to conventional optics. (c) 2025 The Author(s).

We developed a small form factor, microscope objective like, wavefront shaping assisted optical device (Wavelens) to produce and control illumination patterns for use on Light-Sheet Fluorescence Microcopy (LSFM). We characterize it and demonstrate its capability to perform in vivo imaging using fluorescently labeled specimens, outperforming conventional optics.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

We explore wavefront shaping techniques to produce and control illumination patterns for use on Light-Sheet Fluorescence Microcopy (LSFM). We demonstrate the use of these patterns to perform in vivo imaging using fluorescently labeled specimens.

Introduction: This study aimed to evaluate and compare canal transportation and centering ability of 4 different root canal preparation systems produced with thermal treatments by means of micro-computed tomographic imaging. Methods: Eighty mesial canals of human extracted mandibular molars were selected based on similar morphologic parameters and were randomly assigned to 4 experimental groups (n = 20) according to the canal instrumentation technique: HyFlex CM (HCM [Coltene-Whaledent, Allstatten, Switzerland]), HyFlex EDM (HEDM [Coltene-Whaledent]), WaveOne Gold (WOG [Dentsply Sirona, Ballaigues, Switzerland]), and OneCurve (OC [Micro-Mega, Besancon, France]). The specimens were scanned before and after root canal preparation using X-ray micro-computed tomographic imaging at a resolution of 19.9 mm. Apical transportation and centering ability were then analyzed at 3 different levels: 3 mm, 5 mm, and 7 mm from the apex, representing the apical, midroot, and coronal thirds of the root, respectively. One-way analysis of variance and Kruskal-Wallis tests were used to statistically compare the groups. The significance level was set at 5%. Results: HCM caused less canal transportation than WOG at the 3-mm level in both the buccal and lingual canals (P<.05). Also, HCM resulted in less canal transportation than WOG and OC at the 7-mm level regarding lingual canals. No statistically significant differences were recorded between the groups when the mean centering ratios were compared. Conclusions: The 4 evaluated systems safely prepared root canals causing minimal canal transportation and producing relatively centered preparations. In terms of canal transportation, HCM performed better than WOG at the apical level and better than WOG and OC at the coronal level.

Breast cancer and Glioblastoma brain cancer are severe malignancies with poor prognosis. In this study, primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. High resolution imaging of both primary and secondary cancer cell spheroids is possible using a custom multi-angle Light Sheet Fluorescence Microscope. Such a technique is successful in realizing preclinical drug screening, while enables the discrimination among physiologic tumor parameters.

Optical tomography in biomedical imaging is a highly dynamic field in which non-invasive optical and computational techniques are combined to obtain a three dimensional representation of the specimen we are interested to image. Although at optical wavelengths scattering is the main obstacle to reach diffraction limited resolution, recently several studies have shown the possibility to image even objects fully hidden behind a turbid layer exploiting the information contained in the speckle autocorrelation via an iterative phase retrieval algorithm. In this work we explore the possibility of blind three dimensional reconstruction approach based on the Optical Projection Tomography principles, a widely used tool to image almost transparent model organism such as C. Elegans and D. Rerio. By using autocorrelation information rather than projections at each angle we prove, both numerically and experimentally, the possibility to perform exact three dimensional reconstructions via a specifically designed phase retrieval algorithm, extending the capability of the projection-based tomographic methods to image behind scattering curtains. The reconstruction scheme we propose is simple to implement, does not require post-processing data alignment and moreover can be trivially implemented in parallel to fully exploit the computing power offered by modern GPUs, further reducing the need for costly computational resources.

Multispectral imaging and spectroscopic analysis are valuable tools for the study of materials of cultural heritage objects. Accurate registration of spectral images into a spectral cube increases the precision and quality of spectral measurements, supporting portable and low cost multispectral imaging systems.

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy.

Recently great progress has been made in biological and biomedical imaging by combining non-invasive optical methods, novel adaptive light manipulation and computational techniques for intensity-based phase recovery and three dimensional image reconstruction. In particular and in relation to the work presented here, Optical Projection Tomography (OPT) is a well-established technique for imaging mostly transparent absorbing biological models such as C. Elegans and Danio Rerio. On the contrary, scattering layers like the cocoon surrounding the Drosophila during the pupae stage constitutes a challenge for three dimensional imaging through such a complex structure. However, recent studies enabled image reconstruction through scattering curtains up to few transport mean free paths via phase retrieval iterative algorithms allowing to uncover objects hidden behind complex layers. By combining these two techniques we explore the possibility to perform a three dimensional image reconstruction of fluorescent objects embedded between scattering layers without compromising its structural integrity. Dynamical cross correlation registration was implemented for the registration process due to translational and flipping ambiguity of the phase retrieval problem, in order to provide the correct aligned set of data to perform the back-projection reconstruction. We have thus managed to reconstruct a hidden complex object between static scattering curtains and compared with the effective reconstruction to fully understand the process before the in-vivo biological implementation.

Optical Neuroimaging is a highly dynamical field of research owing to the combination of many advanced imaging techniques and computational tools that uncovered unexplored paths through the functioning of the brain. Light propagation modelling through such complicated structures has always played a crucial role as the basis for a high resolution and quantitative imaging where even the slightest improvement could lead to significant results. Fluorescence Diffuse Optical Tomography (fDOT), a widely used technique for three dimensional imaging of small animals and tissues, has been proved to be inaccurate for neuroimaging the mouse head without the knowledge of a-priori anatomical information of the subject. Commonly a normalized Born approximation model is used in fDOT reconstruction based on forward photon propagation using Diffusive Equation (DE) which has strong limitations in the optically clear regime. The presence of the Cerebral Spinal Fluid (CSF) instead, a thin optically clear layer surrounding the brain, can be more accurately taken into account using Monte Carlo approaches which nowadays is becoming more usable thanks to parallelized GPU algorithms. In this work we discuss the results of a synthetic experimental comparison, resulting to the increase of the accuracy for the Born approximation by introducing the CSF layer in a realistic mouse head structure with respect to the current model. We point out the importance of such clear layer for complex geometrical models, while for simple slab phantoms neglecting it does not introduce a significant error.

Diffuse Optical Tomography commonly neglects or assumes as insignificant the presence of optically clear regions in biological tissues, estimating their contribution as a small perturbation to light transport. The inaccuracy introduced by this practice is examined in detail in the context of a complete, based on realistic geometry, virtual fluorescence Diffuse Optical Tomography experiment where a mouse head is imaged in the presence of cerebral spinal fluid. Despite the small thickness of such layer, we point out that an error is introduced when neglecting it from the model with possibly reduction in the accuracy of the reconstruction and localization of the fluorescence distribution within the brain. The results obtained in the extensive study presented here suggest that fluorescence diffuse neuroimaging studies can be improved in terms of quantitative and qualitative reconstruction by accurately taking into account optically transparent regions especially in the cases where the reconstruction is aided by the prior knowledge of the structural geometry of the specimen. Thus, this has only recently become an affordable choice, thanks to novel computation paradigms that allow to run Monte Carlo photon propagation on a simple graphic card, hence speeding up the process a thousand folds compared to CPU-based solutions.

One of the major challenges within Optical Imaging, photon propagation through clear layers embedded between scattering tissues, can be now efficiently modelled in real-time thanks to the Monte Carlo approach based on GPU. Because of its nature, the photon propagation problem can be very easily parallelized and ran on low cost hardware, avoiding the need for expensive Super Computers. A comparison between Diffusion and MC photon propagation theory is presented in this work with application to neuroimaging, investigating low scattering regions in a mouse-like phantom. Regions such as the Cerebral Spinal Fluid, are currently not taken into account in the classical computational models because of the impossibility to accurately simulate light propagation using fast Diffusive Equation approaches, leading to inaccuracies during the reconstruction process. The goal of the study presented here, is to reduce and further improve the computation accuracy of the reconstructed solution in a highly realistic scenario in the case of neuroimaging in preclinical mouse models.

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.

Computational Medicine efforts related to translating Cancer computational models to the clinical practice are focusing on identifying and testing ways to validate the models proposed in vivo before tumor resection. In real life this is actually difficult if not impossible, since patients are treated right away and there is no direct way of imaging the tumor growing. However, in this work, we attempt to validate the simulated outcome of a mathematical tumor growth model of reaction-diffusion type with the actual tumor behavior in human cancer cell lines injected subcutaneously and grown as xenografts in immunodeficient mice by utilizing fluorescence molecular tomography performed in vivo. We show that knowing the initial spatial concentration of the viable cancer cell population, as well as hypoxia and vascularity significantly improves the in silico predictions. Such simulations provide patient specific details that play a significant role in the evolution of the tumor under study.

Optical Projection Tomography and Light Sheet Microscopy have been recent revolutionary techniques for imaging in biological specimens with 3D volume rendering of high resolution. A comparison study is performed on the basic aspects of each modality and combined application on selected specimen is presented. The imaging performance characteristics of the two modalities are calculated and analyzed. Studies in model organisms such as Caenorhabditis Elegans and Parhyale, are shown and the significance of each method is discussed in relation to the specific applications they are best tailored for.

Even though surface morphology is always taken into account when assessing clinically pigmented skin lesions, it is not captured by most modern imaging systems using digital imaging. Our aim is to develop a novel three-dimensional (3D) imaging technique to record detailed information of the surface anatomy of melanocytic lesions that will enable improved classification through digital imaging. The apparatus consists of three high-resolution cameras, a light source, and accompanying software. Volume measurements of specific phantoms using volumetric tubes render slightly lower values than those obtained by our 3D imaging system (mean%± SD, 3.8%± 0.98, P<0.05). To examine the reproducibility of the method, sequential imaging of melanocytic lesions is carried out. The mean%± SD differences of area, major axis length, volume, and maximum height are 2.1%± 1.1, 0.9%± 0.8, 3.8%± 2.9, and 2.5%± 3.5, respectively. Thirty melanocytic lesions are assessed, including common and dysplastic nevi and melanomas. There is a significant difference between nevi and melanomas in terms of variance in height and boundary asymmetry (P<0.001). Moreover, dysplastic nevi have significantly higher variances in pigment density values than common nevi (P<0.001). Preliminary data suggest that our instrument has great potential in the evaluation of the melanocytic lesions. However, these findings should be confirmed in larger-scale studies.

In vivo optical imaging of biological tissue not only requires the development of new theoretical models and experimental procedures, but also the design and construction of realistic tissue-mimicking phantoms. However, most of the phantoms available currently in literature or the market, have either simple geometrical shapes (cubes, slabs, cylinders) or when realistic in shape they use homogeneous approximations of the tissue or animal under investigation. The goal of this study is to develop a non-homogeneous realistic phantom that matches the anatomical geometry and optical characteristics of the mouse head in the visible and near-infrared spectral range. The fabrication of the phantom consisted of three stages. Initially, anatomical information extracted from either mouse head atlases or structural imaging modalities (MRI, XCT) was used to design a digital phantom comprising of the three main layers of the mouse head; the brain, skull and skin. Based on that, initial prototypes were manufactured by using accurate 3D printing, allowing complex objects to be built layer by layer with sub-millimeter resolution. During the second stage the fabrication of individual molds was performed by embedding the prototypes into a rubber-like silicone mixture. In the final stage the detailed phantom was constructed by loading the molds with epoxy resin of controlled optical properties. The optical properties of the resin were regulated by using appropriate quantities of India ink and intralipid. The final phantom consisted of 3 layers, each one with different absorption and scattering coefficient (μa,μs) to simulate the region of the mouse brain, skull and skin.