Imaging of dense cell cultures by multiwavelength lens-free video microscopy.

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.

A new method for x-ray scatter correction: first assessment on a cone-beam CT experimental setup.

Cone-beam computed tomography (CBCT) enables three-dimensional imaging with isotropic resolution and a shorter acquisition time compared to a helical CT scanner. Because a larger object volume is exposed for each projection, scatter levels are much higher than in collimated fan-beam systems, resulting in cupping artifacts, streaks and quantification inaccuracies. In this paper, a general method to correct for scatter in CBCT, without supplementary on-line acquisition, is presented. This method is based on scatter calibration through off-line acquisition combined with on-line analytical transformation based on physical equations, to adapt calibration to the object observed. The method was tested on a PMMA phantom and on an anthropomorphic thorax phantom. The results were validated by comparison to simulation for the PMMA phantom and by comparison to scans obtained on a commercial multi-slice CT scanner for the thorax phantom. Finally, the improvements achieved with the new method were compared to those obtained using a standard beam-stop method. The new method provided results that closely agreed with the simulation and with the conventional CT scanner, eliminating cupping artifacts and significantly improving quantification. Compared to the beam-stop method, lower x-ray doses and shorter acquisition times were needed, both divided by a factor of 9 for the same scatter estimation accuracy.

Dynamics of cell and tissue growth acquired by means of extended field of view lensfree microscopy.

In this paper, we discuss a new methodology based on lensfree imaging to perform wound healing assay with unprecedented statistics. Our video lensfree microscopy setup is a simple device featuring only a CMOS sensor and a semi coherent illumination system. Yet it is a powerful mean for the real-time monitoring of cultivated cells. It presents several key advantages, e.g. integration into standard incubator, compatibility with standard cell culture protocol, simplicity and ease of use. It can perform the follow-up in a large field of view (25 mm(2)) of several crucial parameters during the culture of cells i.e. their motility, their proliferation rate or their death. Consequently the setup can gather large statistics both in space and time. Here we uses this facility in the context of wound healing assay to perform label-free measurements of the velocities of the fronts of proliferation of the cell layer as a function of time by means of particle image velocimetry (PIV) processing. However, for such tissue growth experiments, the field of view of 25 mm(2) remains not sufficient and results can be biased depending on the position of the device with respect to the recipient of the cell culture. Hence, to conduct exhaustive wound healing assays, we propose to enlarge the field of view up to 10 cm(2) through a raster scan, by moving the source/sensor with respect to the Petri dish. We have performed acquisitions of wound healing assay (keratinocytes HaCaT) both in real-time (25 mm(2)) and in final point (10 cm(2)) to assess the combination of velocimetry measurements and final point wide field imaging. In the future, we aim at combining directly our extended field of view acquisitions (>10 cm(2)) with real time ability inside the incubator.

Lensfree diffractive tomography for the imaging of 3D cell cultures.

New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup.

Assessment of vertebral fracture using densitometric morphometry.

Diagnosis of vertebral fracture is critical for management of osteoporosis, as existence of such deformities substantially increases the risk of subsequent fracture. Thus, accurate and precise techniques allowing detection of such deformities are essential to clinicians. So far, this detection has been performed by spinal lateral X-rays. More advanced techniques have recently been developed, based on dual energy X-ray absorptiometry (DXA). This review describes these different techniques and discusses the effectiveness of the DXA technique to assess vertebral deformities compared to X-ray. The use of DXA detection of vertebral fracture for clinical practice and clinical trials is discussed. Specifically, vertebral morphometry using DXA provides an excellent specificity, with moderate sensitivity. The major limitation of the DXA vertebral assessment is the poor quality of images of thoracic vertebrae. The clinical utility of vertebral morphometry using densitometry may help screening patients with vertebral fracture, but technological improvements are necessary to improve image quality.

High-throughput monitoring of major cell functions by means of lensfree video microscopy.

Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.

Bacteria detection with thin wetting film lensless imaging.

Lensless on-chip imaging is a promising technique to count and monitor cells and micro-objects in liquid sample. In this paper we apply this technique to the observation of µL sample containing bacteria evaporated onto a microscope slide. Compared with previously reported techniques, a large improvement in signal to noise ratio is obtained due to the presence of a few µm thick wetting film creating a micro-lens on top of each bacteria. In these conditions, standard CMOS sensor are able to detect micro-objects as small as few µm, e.g. E.coli and Bacillus subtilis bacteria and 1 µm polymer beads with a large signal to noise ratio of 45 ± 10. An overall detection efficiency of 85 ± 7% and a co-localization error of σ(1D) = 1.1µm compared with reference fluorescence microscopy images are achieved. This novel technique will be used as a pre-positioning tool prior to other optical identification methods, e.g. Raman spectroscopy.

Analytical method for localizing a fluorescent inclusion in a turbid medium.

We describe a novel method for localizing a fluorescent inclusion in a homogeneous turbid medium through the use of time-resolved techniques. Based on the calculation of the mean time of the fluorescence curves, the method does not require a priori knowledge of either the fluorescence lifetime or the mean time of the instrument response function since it adopts a differential processing approach. Theoretical expressions were validated and experiments for assessing the accuracy of localization were carried out on liquid optical phantoms with a small fluorescent inclusion. The illumination and detection optical fibers were immersed in the medium to achieve infinite medium geometry as required by the model used. The experimental setup consisted of a time-correlated single-photon counting system. Submillimeter accuracy was achieved for the localization of the inclusion.

Fluorescence diffuse optical tomographic (fDOT) system for small animal studies.

This paper presents a new fluorescence diffuse optical tomographic (fDOT) system and its associated reconstruction method. It is able to reconstruct the fluorescence yield even in heterogeneous and highly attenuating regions. Furthermore our reconstruction method makes mouse inspection without immersion in optical index matching liquid (Intralipid and ink) possible. Some phantom experiments have been carried out to characterize this new fDOT system and to validate its use for heterogeneous media. A mice study consisting in the follow up of the lungs at different stages of tumor development is then related. These results validate the use of our system for biological studies of small animals.

Noncontact fluorescence diffuse optical tomography of heterogeneous media.

Fluorescence-enhanced diffuse optical tomography is expected to be useful to the collection of functional information from small animal models. This technique is currently limited by the extent of tissue heterogeneity and management of the shape of the animals. We propose an approach based on the reconstruction of object heterogeneity, which provides an original solution to the two problems. Three evaluation campaigns are described: the first two were performed on phantoms designed to test the reconstructions in highly heterogeneous media and noncontact geometries; the third was conducted on mice with lung tumors to test fluorescence yield reconstruction feasibility in vivo.

Image resolution and magnification using a cone beam densitometer: optimizing data acquisition for hip morphometric analysis.

Bone mineral density (BMD) is a primary determinant of hip fracture risk. However, other factors, notably the femoral geometry, can influence hip fracture risk. The purpose of this study was to evaluate the potential of a new cone beam densitometer, the DMS Lexxos, in order to visualise femoral morphometry. Resolution, magnification and distortion were assessed in vitro using a line pair test pattern and a matrix test object. Results were given in comparison with currently available systems: the Hologic Discovery A and the Lunar Prodigy densitometers. The DMS Lexxos image resolution was the same in the longitudinal and transversal directions evaluated between 1.4 and 0.5 line pairs/mm (lps/mm) for an attenuation varying from 25 to 325 mm of Perplex. The longitudinal resolution was evaluated between 0.9 and 0.5 lps/mm with the Hologic Discovery densitometer, and inferior to 0.5 with the Lunar Prodigy; as for transversal resolution, it varied from 0.63 to 0.5 lps/mm and from 0.6 to inferior 0.5 lps/mm, respectively. The image was isotropic without magnification with the GE-Lunar Prodigy, whereas there was only a transversal magnification with the Hologic Discovery device. The magnification was about 1.17% cm(-1 )in the two directions, while increasing the distance of the phantom above the examination table with the Lexxos. This magnification was isotropic without distortion. The magnification could be evaluated from two images taken before and after translation of the C-arm, and a magnification correction could be applied. This method was applied to a phantom and to a human cadaver femoral bone.

Total dose incurred by patients and staff from BMD measurement using a new 2D digital bone densitometer.

Dual energy X-ray absorptiometry (DXA) is a widely used and precise technique for non-invasive assessment of bone mineral density. The DXA systems have evolved from pencil X-ray beam (single detector) to fan beam (linear array detector) and recently cone beam densitometers (bi-dimensional detector), allowing for an examination to occur without any scanning and with a short acquisition time. The purpose of this study was to evaluate patient and staff dose from a new cone beam densitometer, the DMS Lexxos. Measurements were performed on a DMS Lexxos bone densitometer prototype. An anthropomorphic phantom and thermoluminescent dosimeters were used to evaluate the effective dose. Ionization chambers and electronic personal dosimeters were used to evaluate the staff dose. The effective dose is 8.4 micro Sv for an anteroposterior spine examination and 4.8 micro Sv for a femoral neck in standard mode. The averaged scattered dose rate (ambient dose equivalent) at 1 m from the beam is evaluated at 226 micro Sv/h. Assuming six patients per hour with two views per patient, the time averaged dose rate is evaluated at 2.9 micro Sv/h. By the personal dosimeter, the staff dose (Hp 10) at 1 m from the beam is evaluated at 0.23 micro Sv per examination. For one examination, patient and staff dose from this new technology remains low: in the same range as the fan-beam densitometer.