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International audience; In this article, we are presenting an original selective plane illumination fluorescence microscope dedicated to image “Organ-on-chip”-like biostructures in microfluidic chips. In order to be able to morphologically analyze volumetric samples in development at the cellular scale inside microfluidic chambers, the setup presents a compromise between relatively large field of view (∼ 200 µm) and moderate resolution (∼ 5 µm). The microscope is based on a simple design, built around the chip and its microfluidic environment to allow 3D imaging inside the chip. In particular, the sample remains horizontally avoiding to disturb the fluidics phenomena. The experimental setup, its optical characterization and the first volumetric images are reported.

The development of vascular networks on-chip is crucial for the long-term culture of three-dimensional cell aggregates such as organoids, spheroids, tumoroids, and tissue explants. Despite the rapid advancement of microvascular network systems and organoid technology, vascularizing organoids-on-chips remains a challenge in tissue engineering. Moreover, most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical settings to operate. Considering these constraints, we developed an innovative platform to establish and monitor the formation of endothelial networks around model spheroids of mesenchymal and endothelial cells as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 15 days on-chip. Importantly, these networks were functional, demonstrating intravascular perfusion within the spheroids or vascular organoids connected to neighbouring endothelial beds. This microphysiological system thus represents a viable organ-on-chip model to vascularize biological tissues and should allow to establish perfusion into organoids using advanced microfluidics.

Dirofilaria immitis causes life-threatening heart disease in dogs, thus screening of dog populations is important. Lens-free technology (LFT) is a low-cost imaging technique based on light diffraction that allows computerized recognition of small objects in holographic images. We evaluated an algorithm capable of recognizing microfilariae in canine whole blood using the LFT. We examined 3 groups of 10 EDTA blood specimens, from dogs with microfilaremia (group A), healthy dogs (B), and dogs with hematologic modifications other than microfilaremia (C). The LFT analyzer photographed repeated series of 5 images of all samples. The algorithm declared a sample positive if a microfilaria was detected on ≥1, ≥2, or ≥3 of the 5 images of a series. Microfilariae were detected visually in the images in 9 of 10 cases in group A; no microfilariae were seen in the images from groups B and C. Of the 30 cases, there were 14, 4, and only 3 false-positives with the 1 of 5, 2 of 5, and 3 of 5 image cutoffs, respectively. There were no false-negatives, regardless of cutoff. LFT seems useful for detecting microfilaria and could have application in clinical pathology.

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 present a light sheet fluorescence microscope dedicated to image “Organ-on-chip”-like biostructures in microfluidic chip. Based on a simple design, the setup is built around the chip and its environment to allow 3D imaging inside the chip in a microfluidic laboratory. The experimental setup, its optical characterization and first volumetric images are reported.

Very wide field of view imaging can be used in biology to infer statistical information on cell populations from a single-shot acquisition. In particular, for applications in hematology, fluorescence wide-field of view imaging could be an alternative to standard fluorescence flow cytometry methods; it can be useful to achieve standard blood tests, such a leukocyte count or leukocyte differential count. We will introduce two 30mm2 wide-field fluorescence imaging set-ups and compare their performances. Both systems achieve 1x magnification. The first one is based on a macro-photography lens. Although such a system optimizes the resolution and field of view, it suffers from its bulkiness. With the second system, we seek miniaturization while loosening the requirements on image quality. It is based on a lens-less approach with a fiber plate optical relay. The potential of the two systems for hematology analyzes will be illustrated with the imaging of labelled white blood cells.

Flow cytometry is the main technology used in hematology analyzers. However, this technology requires bulky and complex hardware systems. Lens-free imaging is an emerging microscopy technique based on a simple and compact inline holography setup. This technique enables to image a large field-of-view (≈30mm²) leading to statistical counting (>10 000 cells) in a single-shot acquisition consistent with performances required in hematology. We report high accuracy platelet count in 54 platelet-rich plasma samples. This accuracy can be achieved through a wise choice of the illumination spectral properties and an optimized algorithmic chain dedicated to small pure phase objects.

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.

Very wide-field of view imaging can provide statistical data on large cell populations in a single acquisition. In this paper, we describe a multimodal imaging system combining brightfield, phase and fluorescence contrasts. Its greater simplicity and lower cost compared to flow cytometry make it suitable for Point-Of-Care applications. The system’s resolution was characterized on calibrated beads and resolution targets. We illustrate the potential of the single-shot imaging approach in hematology by studying the specific morphologies of white blood cell sub-types. The results suggest that very wide field of view imaging could be an alternative to flow cytometry for some applications in hematology.

International audience; Phase and fluorescence are complementary contrasts that are commonly used in biology. However, the coupling of these two modalities is traditionally limited to high magnification and complex imaging systems. For statistical studies of biological populations, a large field-of-view is required. We describe a 30 mm(2) field-of-view dual-modality mesoscope with a 4-mu m resolution. The potential of the system to address biological questions is illustrated on white blood cell numeration in whole blood and multiwavelength imaging of the human osteosarcoma (U2-OS) cells.

The cytology of the cerebrospinal fluid is traditionally performed by an operator (physician, biologist) by means of a conventional light microscope. The operator visually counts the leukocytes (white blood cells) present in a sample of cerebrospinal fluid (10 μl). It is a tedious job and the result is operator-dependent. Here in order to circumvent the limitations of manual counting, we approach the question of numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis by means of lens-free microscopy. In a first step, a prospective counts of leukocytes was performed by five different operators using conventional optical microscopy. The visual counting yielded an overall 16.7p 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 was adapted step-by-step for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes. The optimization of the automatic lens-free counting was based on the prospective analysis of 215 cerebrospinal fluid specimens. The optimized algorithm yielded a 100p sensitivity and a 86p 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 100p sensitivity and a 79p 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; 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.

Lensfree in-line holography permits to image large fields of view with resolution of few micrometers. By adding a fluorescence microscopy detection pathway, we can simultaneously image samples with a better resolution and a complementary contrast.

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.

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.

SPIE Laser Damage - 46th Annual Symposium on Optical Materials for High Power Lasers, National Institute of Standards andTechnology, Boulder, CO, SEP 14-17, 2014; International audience; We present the development of a laser damage and ablation test-bench able to accommodate ultrashort pulses down to 10 fs pulse duration. The laser test-bench is operated in air and we demonstrate its capability to accurately measure the damage and ablation characteristics of optical materials, like fused silica, irradiated by single ultrashort pulses of < 15 fs pulse duration. The careful characterization of beam propagation allows us to ascertain the precise retrieval of laser-induced damage and ablation threshold fluences as well as to identify the energetic regime yielding to beam filamentation.

Reda Kubiliūtė,1,2 Ksenia A Maximova,3 Alireza Lajevardipour,1 Jiawey Yong,1 Jennifer S Hartley,1 Abu SM Mohsin,1 Pierre Blandin,3 James WM Chon,1 Marc Sentis,3 Paul R Stoddart,1 Andrei Kabashin,3 Ričardas Rotomskis,2 Andrew HA Clayton,1,4 Saulius Juodkazis1,4 1Centre for Micro-Photonics and Industrial Research Institute Swinburne, Faculty of Engineering and Industrial Sciences Swinburne University of Technology, Hawthorn, VIC, Australia; 2Laboratory of Biomedical Physics, Vilnius University Institute of Oncology, Baublio, Vilnius, Lithuania; 3Aix-Marseille University, Centre National de la Recherche Scientifique (CNRS), Lasers, Plasmas and Photonics Processing Laboratory, Campus de Luminy, Marseille, France; 4The Australian National Fabrication Facility, Victoria node, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, VIC, Australia Abstract: Aqueous solutions of ultra-pure gold nanoparticles have been prepared by methods of femtosecond laser ablation from a solid target and fragmentation from already formed colloids. Despite the absence of protecting ligands, the solutions could be (1) fairly stable and poly size-dispersed; or (2) very stable and monodispersed, for the two fabrication modalities, respectively. Fluorescence quenching behavior and its intricacies were revealed by fluorescence lifetime imaging microscopy in rhodamine 6G water solution. We show that surface-enhanced Raman scattering of rhodamine 6G on gold nanoparticles can be detected with high fidelity down to micromolar concentrations using the nanoparticles. Application potential of pure gold nanoparticles with polydispersed and nearly monodispersed size distributions are discussed. Keywords: nanotechnologies applications, methods of nanofabrication and processing, materials for nanomedicine

as the Ab42/Ab40 ratio, were increased in Purkinje neurons from sporadic AD cases as compared to controls. However, the levels of Ab42 as well as Ab40 were clearly lower in Purkinje neurons than in pyramidal neurons. Based on the volume of the captured neurons the intraneuronal molar concentrations of Ab were calculated. Conclusions: We suggest that the concentration of intracellular Ab42 correlates to the neuropathology of Alzheimer’s disease.

International audience; We present the generation of a picosecond polarized supercontinuum in highly birefringent multimodal microstructured fiber. The initial steps of the spectral broadening are dominated by intermodal four-wave mixing controlled by the specific fiber design. Using a low repetition rate ultra-stable solid state laser, a pulse train well-suited for versatile time-domain fluorescence lifetime imaging applications is obtained

International audience; We present the instrumental development of a versatile total internal reflection fluorescence lifetime imaging microscopy setup illuminated by a supercontinuum laser source. It enables performing wide- field fluorescence lifetime imaging with subwavelength axial resolution for a large range of fluorophores. The short overall acquisition time and the axial resolution are well suited for dynamic neurobiological applications.

International audience; We developed a Total Internal Reflection Fluorescence Lifetime Imaging Microscope to perform functional imaging of living cells membranes labeled with FRET couples. Förster Resonance Energy Transfer efficiency can thus be followed with subwavelength axial resolution.

We present an experimental and numerical study of supercontinuum generation extended in the visible part of the spectrum by using a selective optical coupling of the pump wave in the largely anomalous dispersion regime. The broadband frequency generation is induced by an initial four-wave mixing process that converts the pump wave at 1064 nm into 831 nm anti-Stokes and 1478 nm Stokes wavelengths. Phase matching is ensured on such a large frequency shift thanks to a microstructured multimodal fiber with a specific design. Continuum generation is therefore enhanced around the two generated sidebands.

International audience; We demonstrate, for the first time, to our knowledge, a diode-pumped passively mode-locked Nd:YVO4 laser, operating on the 4F3/2-4I9/2 transition of the neodymium ion at 914 nm. We obtained 8.8 ps pulses at approximately 914 nm at a repetition rate of 94 MHz, and an averaged output power of 87 mW by using a semiconductor saturable absorber mirror

International audience; An ultrashort laser-assisted method for fast production of concentrated aqueous solutions of ultrapure Si-based colloidal nanoparticles is reported. The method profits from the 3D geometry of femtosecond laser ablation of water-dispersed microscale colloids, prepared preliminarily by the mechanical milling of a Si wafer, in order to avoid strong concentration gradients in the ablated material and provide similar conditions of nanocluster growth within a relatively large laser caustics volume. We demonstrate the possibility for the fast synthesis of non-aggregated, low-size-dispersed, crystalline Si-based nanoparticles, whose size and surface oxidation can be controlled by changing the initial microcolloid concentration and the amount of dissolved oxygen in the water. Due to their much superior purity compared to the chemically synthesized counterparts and their photoluminescence response, the nanoparticles present the possibility for biological in vivo applications such as drug vectoring, imaging, and therapeutics.

We present the development of a time resolved TIRF microscope illuminated by a supercontinuum laser source. It permits to perform wide-field fluorescence lifetime imaging of neurobiological processes at the plasma membrane with subwavelength axial resolution.