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We present a compact 3D diffractive microscope that can be inserted directly in a cell incubator for long-term observation of developing organisms. Our setup is particularly simple and robust, since it does not include any moving parts and is compatible with commercial cell culture containers. It has been designed to image large specimens ( > 100 × 100 × 100 µ m 3 ) with subcellular resolution. The sample’s optical properties [refractive index (RI) and absorption] are reconstructed in 3D from intensity-only images recorded with different illumination angles produced by an LED array. The reconstruction is performed using the beam propagation method embedded inside a deep-learning network where the layers encode the optical properties of the object. This deep neural network is trained for a given multiangle intensity acquisition. After training, the weights of the neural network deliver the 3D distribution of the optical properties of the sample. The effect of spherical aberrations due to the sample holder/air interfaces are taken into account in the forward model. Using this approach, we performed time-lapse 3D imaging of preimplantation mouse embryos over six days. Images of embryos from a single cell (low-scattering regime) to the blastocyst stage (highly scattering regime) were successfully reconstructed. Due to its subcellular resolution, our system can provide quantitative information on the embryos’ development and viability. Hence, this technology opens what we believe to be novel opportunities for 3D label-free live-cell imaging of whole embryos or organoids over long observation times.

SIGNIFICANCE: Multi-laboratory initiatives are essential in performance assessment and standardization-crucial for bringing biophotonics to mature clinical use-to establish protocols and develop reference tissue phantoms that all will allow universal instrument comparison. AIM: The largest multi-laboratory comparison of performance assessment in near-infrared diffuse optics is presented, involving 28 instruments and 12 institutions on a total of eight experiments based on three consolidated protocols (BIP, MEDPHOT, and NEUROPT) as implemented on three kits of tissue phantoms. A total of 20 synthetic indicators were extracted from the dataset, some of them defined here anew. APPROACH: The exercise stems from the Innovative Training Network BitMap funded by the European Commission and expanded to include other European laboratories. A large variety of diffuse optics instruments were considered, based on different approaches (time domain/frequency domain/continuous wave), at various stages of maturity and designed for different applications (e.g., oximetry, spectroscopy, and imaging). RESULTS: This study highlights a substantial difference in hardware performances (e.g., nine decades in responsivity, four decades in dark count rate, and one decade in temporal resolution). Agreement in the estimates of homogeneous optical properties was within 12% of the median value for half of the systems, with a temporal stability of

International audience

Performance assessment and standardization are indispensable for instruments of clinical relevance in general and clinical instrumentation based on photon migration/diffuse optics in particular. In this direction, a multi-laboratory exercise was initiated with the aim of assessing and comparing their performances. 29 diffuse optical instruments belonging to 11 partner institutions of a European level Marie Curie Consortium BitMap1 were considered for this exercise. The enrolled instruments covered different approaches (continuous wave, CW; frequency domain, FD; time domain, TD and spatial frequency domain imaging, SFDI) and applications (e.g. mammography, oximetry, functional imaging, tissue spectroscopy). 10 different tests from 3 well-accepted protocols, namely, the MEDPHOT2 , the BIP3 , and the nEUROPt4 protocols were chosen for the exercise and the necessary phantoms kits were circulated across labs and institutions enrolled in the study. A brief outline of the methodology of the exercise is presented here. Mainly, the design of some of the synthetic descriptors, (single numeric values used to summarize the result of a test and facilitate comparison between instruments) for some of the tests will be discussed.. Future actions of the exercise aim at deploying these measurements onto an open data repository and investigating common analysis tools for the whole dataset.

A lens-free microscope is a simple imaging device performing in-line holographic measurements. In the absence of focusing optics, a reconstruction algorithm is used to retrieve the sample image by solving the inverse problem. This is usually performed by optimization algorithms relying on gradient computation. However the presence of local minima leads to unsatisfactory convergence when phase wrapping errors occur. This is particularly the case in large optical thickness samples, for example cells in suspension and cells undergoing mitosis. To date, the occurrence of phase wrapping errors in the holographic reconstruction limits the application of lens-free microscopy in live cell imaging. To overcome this issue, we propose a novel approach in which the reconstruction alternates between two approaches, an inverse problem optimization and deep learning. The computation starts with a first reconstruction guess of the cell sample image. The result is then fed into a neural network, which is trained to correct phase wrapping errors. The neural network prediction is next used as the initialization of a second and last reconstruction step, which corrects to a certain extent the neural network prediction errors. We demonstrate the applicability of this approach in solving the phase wrapping problem occurring with cells in suspension at large densities. This is a challenging sample that typically cannot be reconstructed without phase wrapping errors, when using inverse problem optimization alone.

We designed a particularly simple, compact and robust microscope for phase and fluorescent imaging. The phase-contrast image is reconstructed from a single, approximately 100 µm defocused image with an algorithm based on a constrained optimization of Fresnel diffraction model. Fluorescence image is recorded in-focus. No mechanical movement of neither sample nor objective or any other part of the system is needed to change between the phase-contrast and fluorescence modality. The change of focus between phase (out-of-focus) and fluorescence (in-focus) imaging is achieved with chromatic aberration specifically enhanced by the optical design of our system. Our microscope is sufficiently compact (10x10x10 cm^3) to fit into a standard biological incubator. The simple and robust design reduces the vibration and the drift of the sample. The absence of motorized components makes the system robust and resistant to the humid conditions inside the biological incubator. These aspects greatly facilitate the long-time observation of cell cultures. We can observe a thousand of cells in parallel in a single field of view (1mm^2) with resolution down to 2 µm. We show FUCCI marked HeLa cell culture observed over three days directly in the incubator. FUCCI (fluorescence ubiquitination cell-cycle indicator), is a genetically encoded, two-colour (red and green), indicator of the progression through the cell cycle: the cells in G1 phase show red fluorescence nuclei while the cells in S, G2 and M phase display green fluorescence within the nuclei. We use phase images for segmentation and tracking of the individual cells which allows us to determine the level of fluorescence in each cell in the green and red fluorescence channel. We compare the obtained statistics with the data from flow cytometer acquired at the end of the observation. We show that we can produce a statistically relevant time-resolved measurement of a cell population while keeping access to the individual cells.

Research is continuously developing imaging methods to better understand the structure and function of biological systems. In this paper, we describe our work to develop lens-free microscopy as a novel mean to observe and quantify cells in 2D and 3D cell culture conditions. At first, we developed a lens-free video microscope based on multiple wavelength acquisitions to perform time-lapse 2D imaging of dense cell culture inside the incubator. We demonstrated that novel phase retrieval techniques enable imaging thin cell samples with high concentration (~15000 cells over a large field of view of 29.4 mm2). The experimental data can next be further analyzed with existing cell profiling and tracking algorithms. As an example, we showed that a 7 days acquisition of a culture of HeLa cells leads to a dataset featuring 2.106 cell point measurements and 104 cell cycle tracks. Recently, we extended our work to the video-microscopy of 3D organoids culture. We showed the capability of lens-free microscopy to perform 3D+time acquisitions of 3D organoids culture. To our knowledge, our technique is the only one able to reconstruct very large volumes of 3D cell culture (~5 mm3) by phase contrast imaging. This new mean of microscopy allowed us to observe a broad range of phenomena present in 3D environments, e.g. self-organizations, displacement of large clusters, merging and interconnection over long distances (>1 mm). In addition, this 3D microscope can capture the interactions of single cells and organoids with their 3D environment, e.g. traction forces generated by large cell aggregates over long distances, up to 1.5 mm. Overall, lens-free microscopy techniques favor ease of use and label-free experimentations as well as time-lapse acquisitions of large datasets. Importantly, we consider that these lens-free microscopy technique can thus expand the repertoire of phenomena that can be studied within 2D and 3D organoids cultures.

Lens-free microscopy aims at recovering sample image from diffraction measurements. The acquisitions are usually processed with an inverse problem approach to retrieve the sample image (phase and absorption). The perfect reconstruction of the sample image is however difficult to achieve. Mostly because of the lack of phase information in the recording process. Recently, deep learning has been used to circumvent this challenge. Convolutional neural networks can be applied to the reconstructed image as a single pass to improve e.g. the signal-to noise ratio or the spatial resolution. Here as an alternative, we propose to alternate between the two classes of algorithms, between the inverse problem approach and the data driven approach. In doing so we intend to improve the reconstruction results but also and importantly try to address the concerns associated with the use of deep learning, namely the generalization and hallucination problems. To demonstrate the applicability of our novel approach we choose to address the case of floating cells sample acquired by means of lens-free microscopy. This is a challenging case with a lot of phase wrapping artifacts that has never been solved using inverse problem approaches only. We demonstrate that our approach is successful in performing the phase unwrapping and that it can next be applied to a very different cell sample, namely the cultures of adherent mammalian cell lines.

They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm2) over a large field of view of 29.4 mm2. Combined with the compactness of the system which fits altogether inside an incubator, lens-free microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry

We propose a simple and compact microscope combining phase imaging with multi-color fluorescence using a standard bright-field objective. The phase image of the sample is reconstructed from a single, approximately 100 μm out-of-focus image taken under semi-coherent illumination, while fluorescence is recorded in-focus in epi-fluorescence geometry. The reproducible changes of the focus are achieved with specifically introduced chromatic aberration in the imaging system. This allows us to move the focal plane simply by changing the imaging wavelength. No mechanical movement of neither sample nor objective or any other part of the setup is therefore required to alternate between the imaging modality. Due to its small size and the absence of motorized components the microscope can easily be used inside a standard biological incubator and allows long-term imaging of cell culture in physiological conditions. A field-of-view of 1.2 mm2 allows simultaneous observation of thousands of cells with micro-meter spatial resolution in phase and multi-channel fluorescence mode. In this manuscript we characterize the system and show a time-lapse of cell culture in phase and multi-channel fluorescence recorded inside an incubator. We believe that the small dimensions, easy usage and low cost of the system make it a useful tool for biological research.

We developed 2D and 3D lens-free microscopes, based on the data processing of raw diffraction images. By nature, these systems allows imaging large field of view of unlabeled samples and easily provide time-lapses.

We have discovered a new 4 h ultradian rhythm that occurs during the interphase of the cell cycle in a wide range of individual mammalian cells, including both primary and transformed cells. The rhythm was detected by holographic lens-free microscopy that follows the histories of the dry mass of thousands of single live cells simultaneously, each at a resolution of five minutes. It was vital that the rhythm was observed in inherently heterogeneous cell populations, thus eliminating synchronization and labeling bias. The rhythm is independent of circadian rhythm, and is temperature-compensated. We show that the amplitude of the fundamental frequency provides a way to quantify the effects of, chemical reagents on cells, thus shedding light on its mechanism. The rhythm is suppressed by proteostasis disruptors and is detected only in proliferating cells, suggesting that it represents a massive degradation and re-synthesis of protein every 4 h in growing cells.

We describe a new ultradian rhythm that occurs during the interphase of the cell cycle in a wide range of mammalian cells, including both primary and transformed cells. The rhythm was detected by holographic lens-free microscopy that follows the individual histories of the dry mass of thousands of live cells simultaneously, each at a resolution of five minutes. Importantly, the rhythm was observed in inherently heterogeneous cell populations, thus eliminating synchronization and labeling bias. The rhythm is independent of circadian rhythm, has a period of 4 hours and is temperature-compensated. We demonstrated that the 4 hr rhythm is suppressed by proteostasis disruptors and is detected only in proliferating cells, suggesting that it represents the periodic dynamics of protein mass in growing cells.Brief teaserWe have revealed a 4 hr rhythm in cell dry mass dynamics that seems to be general in proliferating mammalian cells.

Lens-Free microscopy aims at recovering an observed object such as cell cultures from its diffraction measurements. Diffraction acquisitions are processed with an inverse problem approach to recover optical path difference (OPD) images of the object. Phase unwrapping issue is solved here by using a convolutional neural network (CNN) trained on simulations. The procedure was applied successfully on a neuron cells culture video acquisition.

Quantitative phase imaging (QPI) allows the monitoring of adherent cell cultures continuously over long time periods and it delivers an image of the cell with pixel intensities corresponding to the optical path difference (OPD). These images can be processed to quantify several cellular features. In particular, cell OPD measurements allows the estimation of the cell dry mass, an important metric to study cell mass and growth kinetics. If the ability of QPI to provide phase-contrast images of cells is taken for granted, the accuracy and the precision of QPI cell OPD measurements can still be questioned. Indeed, the reported QPI cell measurements have not yet been assessed with any reference method (e.g. microfluidic resonators). And there is a lack of independent experimental comparison and validation which can hinder the acceptance of QPI in the realms of live-cell mass profiling. With the aim of filling this gap, here we compare three different methods: digital holographic microscopy, quadriwave lateral sheering interferometry and lens-free microscopy (not yet established as a QPI technique). The experimental design is based on the inter-modality comparisons of OPD measurements performed over several tens of cells. To ensure consistency, we performed OPD measurements on a fixed cell culture the same day on the same location. Importantly, the statistical analysis of these measurements allowed us to estimate the precision of QPI cell OPD measurements without any reference material. In addition, we have evaluated the influence of the post-processing steps (baseline subtraction, cell segmentation) on the precision of QPI cell measurements.

Electron tomography is a key technique that enables the visualization of an object in three dimensions with a resolution of about a nanometre. High-quality 3D reconstruction is possible thanks to the latest compressed sensing algorithms and/or better alignment and preprocessing of the 2D projections. Rigid alignment of 2D projections is routine in electron tomography. However, it cannot correct misalignments induced by (i) deformations of the sample due to radiation damage or (ii) drifting of the sample during the acquisition of an image in scanning transmission electron microscope mode. In both cases, those misalignments can give rise to artefacts in the reconstruction. We propose a simple-to-implement non-rigid alignment technique to correct those artefacts. This technique is particularly suited for needle-shaped samples in materials science. It is initiated by a rigid alignment of the projections and it is then followed by several rigid alignments of different parts of the projections. Piecewise linear deformations are applied to each projection to force them to simultaneously satisfy the rigid alignments of the different parts. The efficiency of this technique is demonstrated on three samples, an intermetallic sample with deformation misalignments due to a high electron dose typical to spectroscopic electron tomography, a porous silicon sample with an extremely thin end particularly sensitive to electron beam and another porous silicon sample that was drifting during image acquisitions.

International audience; The structural and functional organization of biological tissues relies on the intricate interplay between chemical and mechanical signaling. Whereas the role of constant and transient mechanical perturbations is generally accepted, several studies recently highlighted the existence of long-range mechanical excitations (i.e., waves) at the supracellular level. Here, we confine epithelial cell monolayers to quasi-onedimensional geometries, to force the establishment of tissue-level waves of well-defined wavelength and period. Numerical simulations based on a self-propelled Voronoi model reproduce the observed waves and exhibit a phase transition between a global and a multinodal wave, controlled by the confinement size. We confirm experimentally the existence of such a phase transition, and show that wavelength and period are independent of the confinement length. Together, these results demonstrate the intrinsic origin of tissue oscillations, which could provide cells with a mechanism to accurately measure distances at the supracellular level.

We present a simple and compact phase imaging microscope for long-term observation of non-absorbing biological samples such as unstained cells in nutritive media. The phase image is obtained from a single defocused image taken with a standard wide-field microscope. Using a semi-coherent light source allows us to computationally re-focus image post-acquisition and recover both phase and transmission of the complex specimen. The simplicity of the system reduces both the cost and its physical size and allows a long-term observation of samples directly in a standard biological incubator. The low cost of the system can contribute to the democratization of science by allowing to perform complex long-term biological experiments to the laboratories with constrained budget. In this proceeding we present several results taken with our prototype and discuss the possibilities and limitations of our system.

We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view (~30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

We introduce a label-free technique based on lens-free microscopy to perform cell counting and cell viability assay. Without the use of any labelling, the discrimination between alive and dead cells is obtained by analyzing the cells on the basis of their holographic signature. We have assessed this novel technique by comparing the obtained results in terms of viability and cell counting against automatic optical counting and trypan blue staining as reference methods. The lensfree measurements agree very well with the reference techniques up to ~30.106 cells/ml. We found a coefficient of determination R2 of 0.99 and a slope of 1.01 for the viability measurements (N=84 CHO cell samples).

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 10(6) cell morphological measurements.

International audience; Lens-free microscopy multispectral acquisitions are processed with an inverse problem approach: a multispectral total variation criterion is defined and minimized with the conjugate gradients method. Reconstruction results show that the method is efficient to recover the phase image of densely packed cells.

International audience; The quantitative phase imaging methods have several advantages when it comes to monitoring cultures of adherent mammalian cells. Because of low photo-toxicity and no need for staining, we can follow cells in a minimally invasive way over a long period of time. The ability to measure the optical path difference in a quantitative manner allows the measurement of the cell dry mass, an important metric for studying the growth kinetics of mammalian cells. Here we present and compare cell measurements obtained with three different techniques: digital holographic microscopy, lens-free microscopy and quadriwave lateral sheering interferometry. We report a linear relationship between optical volume density values measured with these different techniques and estimate the precisions of this measurement for the different individual instruments.

Cerebrospinal fluid cytology is performed by operator-dependant light microscopy as part of the routine laboratory work-flow diagnosis of meningitis. We evaluated operator-independent lens-free microscopy numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis. In a first step, prospective optical microscopy counts of leukocytes done by five different operators yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off. In a second step, the lens-free microscopy algorithm adapted for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes was modified step-by-step in the prospective analysis of 215 cerebrospinal fluid specimens. The definite algorithm yielded a 100% sensitivity and a 86% specificity compared to confirmed diagnostics. In a third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology-confirmed infectious meningitis, yielded a 100% sensitivity and a 79% specificity. Adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories.

International audience; Multicomponent phantom measurements are carried out to evaluate the ability of multispectral time domain diffuse optical tomography in reflectance geometry to quantify the position and the composition of small heterogeneities at depths of 1-1.5 cm in turbid media. Time-resolved data were analyzed with the Mellin-Laplace transform. Results show good localization and correct composition gradation of objects but still a lack of absolute material composition accuracy when no a priori geometry information is known.

International audience; We evaluate the ability of time-resolved diffuse optical tomography to quantify the absorption coefficient of deep objects in turbid media. Phantom experimental results with totally absorbing objects show good quantification up to 0.4 cm-1.

Pathologist examination of tissue slides provides insightful information about a patient’s disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2–4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

International audience; The problem of fluorescence diffuse optical tomography consists in localizing fluorescent markers from near-infrared light measurements. Among the different available acquisition modalities, the time-resolved modality is expected to provide measurements of richer information content. To extract this information, the moments of the time-resolved measurements are often considered. In this paper, a theoretical analysis of the moments of the forward problem in fluorescence diffuse optical tomography is proposed for the infinite medium geometry. The moments are expressed as a function of the source, detector and markers positions as well as the optical properties of the medium and markers. Here, for the first time, an analytical expression holding for any moments order is mathematically derived. In addition, analytical expressions of the mean, variance and covariance of the moments in the presence of noise are given. These expressions are used to demonstrate the increasing sensitivity of moments to noise. Finally, the newly derived expressions are illustrated by means of sensitivity maps. The physical interpretation of the analytical formulae in conjunction with their map representations could provide new insights into the analysis of the information content provided by moments.

International audience; Time-resolved reflectance diffuse optical tomography on phantoms using Silicon photomultipliers (SiPMs) as detectors was carried out. We infer that SiPMs are promising new detectors to probe and accurately quantify biological tissues.

We present the results of a fluorescence tomography experiment showing that fluorescence tomography reconstruction requires a forward model corrected using the optical heterogeneities of the probed medium. We present the procedure we currently use to perform this reconstruction.

Numerical and analytical approaches for the forward solver used in noncontact continuous wave fluorescence optical tomography are considered. Experiments have been carried out on a half cylinder resin phantom with Alexa750 fluorophores inclusions to test the feasibility of these two approaches.

Electron tomography is a key technique that enables the visualization of an object in three dimensions with a resolution of about a nanometre. High-quality 3D reconstruction is possible thanks to the latest compressed sensing algorithms and/or better alignment and preprocessing of the 2D projections. Rigid alignment of 2D projections is routine in electron tomography. However, it cannot correct misalignments induced by (i) deformations of the sample due to radiation damage or (ii) drifting of the sample during the acquisition of an image in scanning transmission electron microscope mode. In both cases, those misalignments can give rise to artefacts in the reconstruction. We propose a simple-to-implement non-rigid alignment technique to correct those artefacts. This technique is particularly suited for needle-shaped samples in materials science. It is initiated by a rigid alignment of the projections and it is then followed by several rigid alignments of different parts of the projections. Piecewise linear deformations are applied to each projection to force them to simultaneously satisfy the rigid alignments of the different parts. The efficiency of this technique is demonstrated on three samples, an intermetallic sample with deformation misalignments due to a high electron dose typical to spectroscopic electron tomography, a porous silicon sample with an extremely thin end particularly sensitive to electron beam and another porous silicon sample that was drifting during image acquisitions.

International audience; Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality as it allows to noninvasively monitor the fluorescence targeted tumors located below the tissue surface. Some drawbacks of this technique are the background fluorescence decreasing the contrast and absorption heterogeneities leading to misinterpretations concerning fluorescence concentrations. We propose a correction technique based on a laser line scanning illumination scheme. We scan the medium with the laser line and acquire at each position of the line both fluorescence and excitation images. We then use the finding that there is a relationship between the excitation intensity profile and the background fluorescence one to predict the amount of signal to subtract to the fluorescence images to get a better contrast. As the light absorption information is contained both in fluorescence and excitation images, this method also permits us to correct the effects of absorption heterogeneities. This technique has been validated on simulations and experimentally. Fluorescent inclusions are observed in several configurations at depths ranging from 1 mm to 1 cm. Results obtained with this technique are compared to those obtained with a classical wide-field detection scheme for the contrast enhancement and to the fluorescence by excitation ratio approach for the absorption correction.

International audience; The noninvasive assessment of flap viability in autologous reconstruction surgery is still an unmet clinical need. To cope with this problem, we developed a proof-of-principle fully automatized setup for fast time-gated diffuse optical tomography exploiting Mellin–Laplace transform to obtain three-dimensional tomographic reconstructions of oxy- and deoxy-hemoglobin concentrations. We applied this method to perform preclinical tests on rats inducing total venous occlusion in the cutaneous abdominal flaps. Notwithstanding the use of just four source-detector couples, we could detect a spatially localized increase of deoxyhemoglobin following the occlusion (up to 550 μM in 54 min). Such capability to image spatio-temporal evolution of blood perfusion is a key issue for the noninvasive monitoring of flap viability.

We developed a new imaging tool that can help pathologists in recording wide-field images of tissue slides. We present a simple cost-effective lensfree imaging method to record 2‒4µm resolution wide-field (10 mm² ‒ 6 cm²) images of stained and unstained tissue slides. To our knowledge, our method is the first technique that enables fast (less than 5 minutes) wide-field lensfree imaging of such dense samples. Multiple holograms are recorded with different wavelength illumination, and a multispectral algorithm is used to retrieve both amplitude and phase. Our method can be used to retrieve images of stained tissue slides. For such absorbing object, the useful information is included in the modulus of the reconstructed complex field. Our method can also be applied to retrieve images of unstained tissue slides, where the useful information is in the retrieved phase. This technique is much cheaper and compact than a conventional microscope and could be made portable. Moreover, it enables wide field unstained tissue slides imaging, which could quickly provide useful information, for example on frozen section biopsies, when a rapid diagnosis is needed during surgery.

An automatic procedure for electron tomography is presented. This procedure is adapted for specimens that can be fashioned into a needle-shaped sample and has been evaluated on inorganic samples. It consists of self-adapting denoising, automatic and accurate alignment including detection and correction of tilt axis, and 3D reconstruction. We propose the exploitation of a large amount of information of an electron tomography acquisition to achieve robust and automatic mixed Poisson-Gaussian noise parameter estimation and denoising using undecimated wavelet transforms. The alignment is made by mixing three techniques, namely (i) cross-correlations between neighboring projections, (ii) common line algorithm to get a precise shift correction in the direction of the tilt axis and (iii) intermediate reconstructions to precisely determine the tilt axis and shift correction in the direction perpendicular to that axis. Mixing alignment techniques turns out to be very efficient and fast. Significant improvements are highlighted in both simulations and real data reconstructions of porous silicon in high angle annular dark field mode and agglomerated silver nanoparticles in incoherent bright field mode. 3D reconstructions obtained with minimal user-intervention present fewer artefacts and less noise, which permits easier and more reliable segmentation and quantitative analysis. After careful sample preparation and data acquisition, the denoising procedure, alignment and reconstruction can be achieved within an hour for a 3D volume of about a hundred million voxels, which is a step toward a more routine use of electron tomography.

We compare mammography breast density (BD(MD)) to the measure of breast composition using a clinical dual energy absorptiometry (DXA) system (BD(DXA)) calibrated to measure breast density. A DXA scanning protocol was developed to scan breasts isolated in the DXA scan field in either a prone pendulous or decubitus mediolateral position. A total of 17 participants were recruited among women undergoing clinical mammography examinations. Each participant had duplicate DXA scans and duplicate craniocaudal-view mammograms of their right breast with repositioning between each scan and one DXA and one craniocaudal-view mammogram of their left breast. The in vivo repeatability (RMS SD) of BD(DXA) and BD(MD) on duplicate scans was found to be 1.2% for BD(DXA) and 1.4% for BD(MD) when repeat BD(MD) measures were made on the same day. When repeat BD(MD) measures of the same breast were made more than 50 days apart, the repeatability decreased to 5.5%. Left and right breast measurements were highly correlated with both techniques at r2 = 0.98 for BD(DXA) and r2 = 0.86 for BD(MD). Moderate correlation (r2 = 0.52) was found between BD(DXA) and BD(MD) measurements. However, after recalibrating the DXA system to mammography reference materials, negative percent fibroglandular values were measured for the most fatty breasts. Thus, our results are reproducible and accurate to common mammography tissue standards, but did not accurately reflect true percent fibroglandular levels and further development of phantom standards are necessary. We conclude that breast composition can be precisely evaluated and assessed with clinical DXA densitometers at a lower dose than with mammographic breast density methods.