Comparative study of fully three-dimensional reconstruction algorithms for lens-free microscopy.

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multiangle acquisitions on 3D cell cultures embedded in extracellular matrices. Lens-free microscopy acquisitions present some inherent issues such as the lack of phase information on the sensor plane and a limited angular coverage. We developed and compared three different algorithms based on the Fourier diffraction theorem to obtain fully 3D reconstructions. These algorithms present an increasing complexity associated with a better reconstruction quality. Two of them are based on a regularized inverse problem approach. To compare the reconstruction methods in terms of artefact reduction, signal-to-noise ratio, and computation time, we tested them on two experimental datasets: an endothelial cell culture and a prostate cell culture grown in a 3D extracellular matrix with large reconstructed volumes up to ∼5 mm3 with a resolution sufficient to resolve isolated single cells. The lens-free reconstructions compare well with standard microscopy.

Time-domain diffuse optical tomography processing by using the Mellin-Laplace transform.

We investigate the use of the Mellin-Laplace transform for reconstructing optical parameters from time-resolved optical tomography in diffusive media. We present here its definition, its mathematical properties, and its sensitivity to variations of optical properties. The method was validated on two-dimensional reconstructions from simulation in the reflection geometry. We conclude that reconstructions based on the Mellin-Laplace transform are more robust to noise than the methods using first moments.

Contact, high-resolution spatial diffuse reflectance imaging system for skin condition diagnosis: a first-in-human clinical trial.

SIGNIFICANCE: Oxygenation is one of the skin tissue physiological properties to follow for patient care management. Furthermore, long-term monitoring of such parameters is needed at the patient bed as well as outside the hospital. Diffuse reflectance spectroscopy has been widely used for this purpose. AIM: The aim of the study is to propose a low-cost system for the long-term measurement of skin physiological parameters in contact. APPROACH: We have developed a low-cost, wearable, CMOS-based device. We propose an original method for processing diffuse reflectance data to calculate the tissue oxygen saturation (StO2). RESULTS: We tested the device for the assessment of tissue oxygenation during a first-in-human clinical trial that took place at the Grenoble University Hospital France. CONCLUSIONS: The results of this clinical trial show a good accordance between our sensor and commercial devices used a reference.

Real time optical method for localization of inclusions embedded in turbid media.

A simple and fast time-domain method for localizing inclusions, fluorescent optical probes or absorbers, is presented. The method offers new possibilities for situations where complete tomographic measurements are not permitted by the examined object, for example in endoscopic examination of the human prostate or the oesophagus. Feasibility has been envisioned with a phantom study conducted on a point-like fluorochrome embedded in a diffusing medium mimicking the optical properties of biological tissues.

A time-domain wavelet-based approach for fluorescence diffuse optical tomography.

PURPOSE: In the context of fluorescence diffuse optical tomography, determining the optimal way to exploit the time-resolved information has been receiving much attention and different features of the time-resolved signals have been introduced. In this article, the authors revisit and generalize the notion of feature, considering the projection of the measurements onto some basis functions. This leads the authors to propose a novel approach based on the wavelet transform of the measurements. METHODS: A comparative study between the reconstructions obtained from the proposed wavelet-based approach and the reconstructions obtained from the reference temporal moments is provided. An inhomogeneous cubic medium is considered. Reconstructions are performed from synthetic measurements assuming Poisson noise statistics. In order to provide fairly comparable reconstructions, the reconstruction scheme is associated with a particular procedure for selecting the regularization parameter. RESULTS: In the noise-free case, the reconstruction quality is shown to be mainly driven by the number of selected features. In the presence of noise, however, the reconstruction quality depends on the type of the features. In this case, the wavelet approach is shown to outperform the moment approach. While the optimal time-resolved reconstruction quality, which is obtained considering the whole set of time samples, is recovered using only eight wavelet functions, it cannot be attained using moments. It is finally observed that the time-resolved information is of limited utility, in terms of reconstruction, when the maximum number of detected photons is lower than 10(5). CONCLUSIONS: The wavelet approach allows for better exploiting the time-resolved information, especially when the number of detected photons is low. However, when the number of detected photons decreases below a certain threshold, the time-resolved information itself is shown to be of limited utility.

Chi2 analysis for estimating the accuracy of optical properties derived from time resolved diffuse-reflectance.

Weighted residuals and the reduced chi(2) (chi(R) (2)) value are investigated with regard to their relevance for assessing optical property estimates using the diffusion equation for time-resolved measurements in turbid media. It is shown and explained, for all photon counting experiments including lifetime estimation, why chi(R) (2) increases linearly with the number of photons when there is a model bias. Only when a sufficient number of photons has been acquired, chi(R) (2) is a pertinent value for assessing the accuracy of mu(a) and mu(s)' estimates. It was concluded that chi(R) (2) is of particular interest for cases of small interfiber separation, low-level scattering, strong absorption and incorrect measurement of instrument response function. It was also found that chi(R) (2) is less pertinent for judging mu(a) in case of air boundary effects.

Application of a wavelet-Galerkin method to the forward problem resolution in fluorescence diffuse optical tomography.

Fluorescence diffuse optical tomography is a powerful tool for the investigation of molecular events in studies for new therapeutic developments. Here, the emphasis is put on the mathematical problem of tomography, which can be formulated in terms of an estimation of physical parameters appearing as a set of Partial Differential Equations (PDEs). The standard polynomial Finite Element Method (FEM) is a method of choice to solve the diffusion equation because it has no restriction in terms of neither the geometry nor the homogeneity of the system, but it is time consuming. In order to speed up computation time, this paper proposes an alternative numerical model, describing the diffusion operator in orthonormal basis of compactly supported wavelets. The discretization of the PDEs yields to matrices which are easily computed from derivative wavelet product integrals. Due to the shape of the wavelet basis, the studied domain is included in a regular fictitious domain. A validation study and a comparison with the standard FEM are conducted on synthetic data.

Data acquisition analysis for femoral morphometric X-ray absorptiometry.

Bone mineral density (BMD) is a contributing factor of hip fracture risk. Other factors, such as lifestyle, the propensity for falls, and femoral geometry may influence the risk of hip fracture. The DMS Lexxos densitometer, a dual-energy X-ray densitometer can produce either a single-energy X-ray or a BMD image. The purpose of this study was to evaluate which of these 2 images enables the best detection to make femoral morphometry measurements. Spatial resolution, contrast, and noise were evaluated separately. A contrast-detail phantom was also used for the purpose of overall visual analysis. The spatial resolution was the same in the 2 images. The contrast was better with the BMD image, but the noise was higher. Using the contrast-detail phantom, the single-energy X-ray image allowed globally a better detection of the objects, but results were in the same range with high contrast values. Hip volunteers' morphometric measurements and the Singh Index were evaluated 3 times for each image by 3 observers, and the intra-, inter-, and global reproducibility was computed. The reproducibility of the measurements seems to be better with the single-energy X-ray image but results were not statistically significantly different. These results suggest that even if the image-quality indices were different, the single-energy X-ray image and BMD image are closely useful for clinical morphometric femoral evaluation.

Model reduction using wavelet multiresolution technique applied to fluorescence diffuse optical tomography.

Fluorescence diffuse optical tomography is a powerful tool for the investigation of molecular events in studies for new therapeutic developments. Here, the stress is put on the mathematical problem of tomography, which can be formulated in terms of an estimation of physical parameters appearing as a set of partial differential equations and solved by the finite element method. This method is well known to be time consuming, and our principal objective is to reduce the model in order to speed up computation. A method based on a wavelet multiresolution technique is presented in detail. A validation study was conducted on synthetic data and experiments.

Optical calibration protocol for an x-ray and optical multimodality tomography system dedicated to small-animal examination.

A small-animal multimodality tomography system dedicated to the coregistration of fluorescence optical signal and x-ray measurements has been developed in our laboratory. The purpose of such a system is to offer the possibility of getting in vivo anatomical and functional information simultaneously. Moreover, anatomical measurements can be used as a regularization factor to achieve more accurate reconstructions of the biodistribution of fluorochromes and to speed up treatment. A dedicated acquisition protocol has been established, and the methodology of the reconstruction of the three-dimensional distribution of the biomarkers under cylindrical geometry consistent with classic computed tomography has been implemented. A phantom study was conducted to evaluate and to fix the parameters for the coregistration. These test experiments were reproduced by considering anesthetized mice that had thin glass tubes containing fluorochromes inserted into their esophagus. The instrument is also used for an in vivo biological study conducted on mice with lung tumors, tagged with near-infrared optical probes (targeting probes such as Transferin-AlexaFluor750).

In vivo mice lung tumor follow-up with fluorescence diffuse optical tomography.

We present in vivo experiments conducted with a new fluorescence diffuse optical tomographic (fDOT) system on cancerous mice bearing mammary murine tumors. We first briefly present this new system that has been developed and its associated reconstruction method. Its main specificity is its ability to reconstruct the fluorescence yield even in heterogeneous and highly attenuating body regions such as lungs and to enable mouse inspection without immersion in optical index matching liquid (Intralipid and ink). Some phantom experiments validate the performance of this new system for heterogeneous media inspection. Its use for a mice study is then related. It consists in the follow-up of the lungs at different stages of tumor development after injection of RAFT-(cRGD)4-Alexa700. As expected, the reconstructed fluorescence increases along with the tumor stage. These results validate the use of our system for biological studies of small animals.

Validation of optical properties quantification with a dual-step technique for biological tissue analysis.

To approach wide-field optical properties quantification in real heterogeneous biological tissue, we developed a Dual-Step setup that couples a punctual diffuse reflectance spectroscopy (DRS) technique with multispectral imaging (MSI). The setup achieves wide-field optical properties assessment through an initial estimation of scattering with DRS, which is used to estimate absorption with MSI. The absolute quantification of optical properties is based on the ACA-Pro algorithm that has been adapted both for DRS and for MSI. This paper validates the Dual-Step system not only on homogeneous Intralipid phantoms but also on a heterogeneous gelatine phantom with different scattering and absorbing properties.

Quantitative comparison of PZT and CMUT probes for photoacoustic imaging: Experimental validation.

Photoacoustic (PA) signals are short ultrasound (US) pulses typically characterized by a single-cycle shape, often referred to as N-shape. The spectral content of such wideband signals ranges from a few hundred kilohertz to several tens of megahertz. Typical reception frequency responses of classical piezoelectric US imaging transducers, based on PZT technology, are not sufficiently broadband to fully preserve the entire information contained in PA signals, which are then filtered, thus limiting PA imaging performance. Capacitive micromachined ultrasonic transducers (CMUT) are rapidly emerging as a valid alternative to conventional PZT transducers in several medical ultrasound imaging applications. As compared to PZT transducers, CMUTs exhibit both higher sensitivity and significantly broader frequency response in reception, making their use attractive in PA imaging applications. This paper explores the advantages of the CMUT larger bandwidth in PA imaging by carrying out an experimental comparative study using various CMUT and PZT probes from different research laboratories and manufacturers. PA acquisitions are performed on a suture wire and on several home-made bimodal phantoms with both PZT and CMUT probes. Three criteria, based on the evaluation of pure receive impulse response, signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) respectively, have been used for a quantitative comparison of imaging results. The measured fractional bandwidths of the CMUT arrays are larger compared to PZT probes. Moreover, both SNR and CNR are enhanced by at least 6 dB with CMUT technology. This work highlights the potential of CMUT technology for PA imaging through qualitative and quantitative parameters.

Cerebrospinal fluid lens-free microscopy: a new tool for the laboratory diagnosis of meningitis.

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.

Chromophore decomposition in multispectral time-resolved diffuse optical tomography.

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.

Relevance of 2D radiographic texture analysis for the assessment of 3D bone micro-architecture.

Although the diagnosis of osteoporosis is mainly based on dual x-ray absorptiometry, it has been shown that trabecular bone micro-architecture is also an important factor in regard to fracture risk. In vivo, techniques based on high-resolution x-ray radiography associated to texture analysis have been proposed to investigate bone micro-architecture, but their relevance for giving pertinent 3D information is unclear. Thirty-three calcaneus and femoral neck bone samples including the cortical shells (diameter: 14 mm, height: 30-40 mm) were imaged using 3D-synchrotron x-ray micro-CT at the ESRF. The 3D reconstructed images with a cubic voxel size of 15 microm were further used for two purposes: (1) quantification of three-dimensional trabecular bone micro-architecture, (2) simulation of realistic x-ray radiographs under different acquisition conditions. The simulated x-ray radiographs were then analyzed using a large variety of texture analysis methods (co-occurrence, spectral density, fractal, morphology, etc.). The range of micro-architecture parameters was in agreement with previous studies and rather large, suggesting that the population was representative. More than 350 texture parameters were tested. A small number of them were selected based on their correlation to micro-architectural morphometric parameters. Using this subset of texture parameters, multiple regression allowed one to predict up to 93% of the variance of micro-architecture parameters using three texture features. 2D texture features predicting 3D micro-architecture parameters other than BV/TV were identified. The methodology proposed for evaluating the relationships between 3D micro-architecture and 2D texture parameters may also be used for optimizing the conditions for radiographic imaging. Further work will include the application of the method to physical radiographs. In the future, this approach could be used in combination with DXA to refine osteoporosis diagnosis.

A study on identification of bacteria in environmental samples using single-cell Raman spectroscopy: feasibility and reference libraries.

We report on our recent efforts towards identifying bacteria in environmental samples by means of Raman spectroscopy. We established a database of Raman spectra from bacteria submitted to various environmental conditions. This dataset was used to verify that Raman typing is possible from measurements performed in non-ideal conditions. Starting from the same dataset, we then varied the phenotype and matrix diversity content included in the reference library used to train the statistical model. The results show that it is possible to obtain models with an extended coverage of spectral variabilities, compared to environment-specific models trained on spectra from a restricted set of conditions. Broad coverage models are desirable for environmental samples since the exact conditions of the bacteria cannot be controlled.

ACA-Pro: calibration protocol for quantitative diffuse reflectance spectroscopy. Validation on contact and noncontact probe- and CCD-based systems.

We have developed an adaptive calibration algorithm and protocol (ACA-Pro) that corrects from the instrumental response of various spatially resolved diffuse reflectance spectroscopy (DRSsr) systems to enable the quantification of absorption and scattering properties based on a Monte Carlo-based look-up-table approach. The protocol involves the use of a calibration reference base built with measurements of a range of different diffusive intralipid phantoms. Moreover, an advanced strategy was established to take into account the experimental variations with an additional measurement of a common solid material, allowing the use of a single calibration reference base for all experiments. The ACA-Pro is validated in contact and noncontact probe-based DRSsr systems. Furthermore, the first results of a setup replacing the probe with a CCD detector are shown to confirm the robustness of the approach.

Quantification in time-domain diffuse optical tomography using Mellin-Laplace transforms.

Simulations and phantom measurements are used to evaluate the ability of time-domain diffuse optical tomography using Mellin-Laplace transforms to quantify the absorption perturbation of centimetric objects immersed at depth 1-2 cm in turbid media. We find that the estimated absorption coefficient varies almost linearly with the absorption change in the range of 0-0.15 cm-1 but is underestimated by a factor that depends on the inclusion depth (~2, 3 and 6 for depths of 1.0, 1.5 and 2.0 cm respectively). For larger absorption changes, the variation is sublinear with ~20% decrease for 뫵a = 0.37 cm-1. By contrast, constraining the absorption change to the actual volume of the inclusion may considerably improve the accuracy and linearity of the reconstructed absorption.

Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.