Multi-laboratory performance assessment of diffuse optics instruments: the BitMap exercise.

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 <5 % over 1 h, and day-to-day reproducibility of <3 % . Other tests encompassed linearity, crosstalk, uncertainty, and detection of optical inhomogeneities. CONCLUSIONS: This extensive multi-laboratory exercise provides a detailed assessment of near-infrared Diffuse optical instruments and can be used for reference grading. The dataset-available soon in an open data repository-can be evaluated in multiple ways, for instance, to compare different analysis tools or study the impact of hardware implementations.

3D time-lapse imaging of a mouse embryo using intensity diffraction tomography embedded inside a deep learning framework.

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 (>100×100×100µm3) 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.

A new ultradian rhythm in mammalian cell dry mass observed by holography.

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.

Real-Time Dual-Wavelength Time-Resolved Diffuse Optical Tomography System for Functional Brain Imaging Based on Probe-Hosted Silicon Photomultipliers.

Near-infrared diffuse optical tomography is a non-invasive photonics-based imaging technology suited to functional brain imaging applications. Recent developments have proved that it is possible to build a compact time-domain diffuse optical tomography system based on silicon photomultipliers (SiPM) detectors. The system presented in this paper was equipped with the same eight SiPM probe-hosted detectors, but was upgraded with six injection fibers to shine the sample at several points. Moreover, an automatic switch was included enabling a complete measurement to be performed in less than one second. Further, the system was provided with a dual-wavelength ( 670 n m and 820 n m ) light source to quantify the oxy- and deoxy-hemoglobin concentration evolution in the tissue. This novel system was challenged against a solid phantom experiment, and two in-vivo tests, namely arm occlusion and motor cortex brain activation. The results show that the tomographic system makes it possible to follow the evolution of brain activation over time with a 1 s -resolution.

Phase and fluorescence imaging with a surprisingly simple microscope based on chromatic aberration.

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.

Graph- and finite element-based total variation models for the inverse problem in diffuse optical tomography.

Total variation (TV) is a powerful regularization method that has been widely applied in different imaging applications, but is difficult to apply to diffuse optical tomography (DOT) image reconstruction (inverse problem) due to unstructured discretization of complex geometries, non-linearity of the data fitting and regularization terms, and non-differentiability of the regularization term. We develop several approaches to overcome these difficulties by: i) defining discrete differential operators for TV regularization using both finite element and graph representations; ii) developing an optimization algorithm based on the alternating direction method of multipliers (ADMM) for the non-differentiable and non-linear minimization problem; iii) investigating isotropic and anisotropic variants of TV regularization, and comparing their finite element (FEM)- and graph-based implementations. These approaches are evaluated on experiments on simulated data and real data acquired from a tissue phantom. Our results show that both FEM and graph-based TV regularization is able to accurately reconstruct both sparse and non-sparse distributions without the over-smoothing effect of Tikhonov regularization and the over-sparsifying effect of L1 regularization. The graph representation was found to out-perform the FEM method for low-resolution meshes, and the FEM method was found to be more accurate for high-resolution meshes.

Confinement-Induced Transition between Wavelike Collective Cell Migration Modes.

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-one-dimensional 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.

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture.

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 106 cell morphological measurements.

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.

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.

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.

Time-domain diffuse optical tomography using silicon photomultipliers: feasibility study.

Silicon photomultipliers (SiPMs) have been very recently introduced as the most promising detectors in the field of diffuse optics, in particular due to the inherent low cost and large active area. We also demonstrate the suitability of SiPMs for time-domain diffuse optical tomography (DOT). The study is based on both simulations and experimental measurements. Results clearly show excellent performances in terms of spatial localization of an absorbing perturbation, thus opening the way to the use of SiPMs for DOT, with the possibility to conceive a new generation of low-cost and reliable multichannel tomographic systems.

Non-rigid alignment in electron tomography in materials science.

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.

Toward noninvasive assessment of flap viability with time-resolved diffuse optical tomography: a preclinical test on rats.

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.

Self-adapting denoising, alignment and reconstruction in electron tomography in materials science.

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.

Laser line illumination scheme allowing the reduction of background signal and the correction of absorption heterogeneities effects for fluorescence reflectance imaging.

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 from 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 with those obtained with a classical wide-field detection scheme for contrast enhancement and with the fluorescence by an excitation ratio approach for absorption correction.

Spatial resolution in depth for time-resolved diffuse optical tomography using short source-detector separations.

Diffuse optical tomography for medical applications can require probes with small dimensions involving short source-detector separations. Even though this configuration is seen at first as a constraint due to the challenge of depth sensitivity, we show here that it can potentially be an asset for spatial resolution in depth. By comparing two fiber optic probes on a test object, we first show with simulations that short source-detector separations improve the spatial resolution down to a limit depth. We then confirm these results in an experimental study with a state-of-the-art setup involving a fast-gated single-photon avalanche diode allowing maximum depth sensitivity. We conclude that short source-detector separations are an option to consider for the design of probes so as to improve image quality for diffuse optical tomography in reflectance.

Depth-enhanced fluorescence imaging using masked detection of structured illumination.

There is a growing interest in imaging fluorescence contrast at depth within living tissues over wide fields of view and in real time. Most methods used to date to improve depth detection of fluorescence information involve acquisition of multiple images, postprocessing of the data using a light propagation model, and are capable of providing either depth-sectioned or tomographic fluorescence information. We introduce a method, termed masked detection of structured illumination, that allows the enhancement of fluorescence imaging at depth without postprocessing. This method relies on the scanning of a collimated beam onto a turbid medium and the physical masking of the point spread function on the detection arm before acquisition on a CCD camera. By preferentially collecting diffuse photons at a chosen source-detector range, this method enhances fluorescence information at depth and has the potential to form images without postprocessing and in real time.

Laser line scanning for fluorescence reflectance imaging: a phantom study and in vivo validation of the enhancement of contrast and resolution.

Intraoperative fluorescence imaging in reflectance geometry is an attractive imaging modality to noninvasively monitor fluorescence-targeted tumors. In some situations, this kind of imaging suffers from poor resolution due to the diffusive nature of photons in tissue. The objective of the proposed technique is to tackle this limitation. It relies on the scanning of the medium with a laser line illumination and the acquisition of images at each position of excitation. The detection scheme proposed takes advantage of the stack of images acquired to enhance the resolution and the contrast of the final image. The experimental protocol is described to fully understand why we overpass the classical limits and validate the scheme on tissue-like phantoms and in vivo with a preliminary testing. The results are compared with those obtained with a classical wide-field illumination.

Time-resolved diffuse optical tomography using fast-gated single-photon avalanche diodes.

We present the first experimental results of reflectance Diffuse Optical Tomography (DOT) performed with a fast-gated single-photon avalanche diode (SPAD) coupled to a time-correlated single-photon counting system. The Mellin-Laplace transform was employed to process time-resolved data. We compare the performances of the SPAD operated in the gated mode vs. the non-gated mode for the detection and localization of an absorbing inclusion deeply embedded in a turbid medium for 5 and 15 mm interfiber distances. We demonstrate that, for a given acquisition time, the gated mode enables the detection and better localization of deeper absorbing inclusions than the non-gated mode. These results obtained on phantoms demonstrate the efficacy of time-resolved DOT at small interfiber distances. By achieving depth sensitivity with limited acquisition times, the gated mode increases the relevance of reflectance DOT at small interfiber distance for clinical applications.