Arkitekt: streaming analysis and real-time workflows for microscopy.

Quantitative microscopy workflows have evolved dramatically over the past years, progressively becoming more complex with the emergence of deep learning. Long-standing challenges such as three-dimensional segmentation of complex microscopy data can finally be addressed, and new imaging modalities are breaking records in both resolution and acquisition speed, generating gigabytes if not terabytes of data per day. With this shift in bioimage workflows comes an increasing need for efficient orchestration and data management, necessitating multitool interoperability and the ability to span dedicated computing resources.

Degradation of Proteoglycans and Collagen in Equine Meniscal Tissues.

Investigate meniscal extracellular matrix degradation. Equine menisci ( = 34 from 17 horses) were studied. Site-matched sections were cut and scored from three regions (ROIs; = 102) and stained for histology, proteoglycan (safranin O and fast green), aggrecan, and collagen cleavage (NITEGE, DIPEN, and C1,2C antibodies, respectively).

Impact of a tilted coverslip on two-photon and STED microscopy.

The advent of super-resolution microscopy has opened up new avenues to unveil brain structures with unprecedented spatial resolution in the living state. Yet, its application to live animals remains a genuine challenge. Getting optical access to the brain requires the use of a 'cranial window', whose mounting greatly influences image quality.

Shadow imaging for panoptical visualization of brain tissue in vivo.

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale.

The Impact of Chemical Fixation on the Microanatomy of Mouse Organotypic Hippocampal Slices.

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy (EM). However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles, and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts, and knowing how to avoid them, takes on special urgency.

Imaging dendritic spines in the hippocampus of a living mouse by 3D-stimulated emission depletion microscopy.

Significance: Stimulated emission depletion (STED) microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples, such as cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging.

Aim: In previous work, we established chronic STED imaging in the hippocampus but the gain in spatial resolution was restricted to the lateral plane.

Second harmonic generation microscopy: a powerful tool for bio-imaging.

Second harmonic generation (SHG) microscopy is an important optical imaging technique in a variety of applications. This article describes the history and physical principles of SHG microscopy and its more advanced variants, as well as their strengths and weaknesses in biomedical applications. It also provides an overview of SHG and advanced SHG imaging in neuroscience and microtubule imaging and how these methods can aid in understanding microtubule formation, structuration, and involvement in neuronal function.

Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue.

Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. The aim of our work was to develop and validate a method based on adaptive optics to achieve an correction of spherical aberrations as a function of imaging depth.

Super-resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge.

The extracellular space (ECS) plays a central role in brain physiology, shaping the time course and spread of neurochemicals, ions, and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brain's parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS.

Combining tensile testing and structural analysis at the single collagen fibril level.

Tensile testing to failure followed by imaging is a simple way of studying the structure-function relationship of connective tissues such as skin, tendon, and ligament. However, interpretation of these datasets is complex due to the hierarchical structures of the tissues spanning six or more orders of magnitude in length scale. Here we present a dataset obtained through the same scheme at the single collagen fibril level, the fundamental tensile element of load-bearing tissues.

Effect of refractive index mismatch on forward-to-backward ratios in SHG imaging.

Nonlinear optical imaging in the epi-direction is used to image subresolution features. We find that a refractive index mismatch between the object to be imaged and the background medium can change the far-field intensity image. As an example, we study second harmonic generation (SHG) microscopy where the forward-to-backward (F/B) ratio is used to quantify subresolution features.

Chronic 2P-STED imaging reveals high turnover of dendritic spines in the hippocampus in vivo.

Rewiring neural circuits by the formation and elimination of synapses is thought to be a key cellular mechanism of learning and memory in the mammalian brain. Dendritic spines are the postsynaptic structural component of excitatory synapses, and their experience-dependent plasticity has been extensively studied in mouse superficial cortex using two-photon microscopy in vivo. By contrast, very little is known about spine plasticity in the hippocampus, which is the archetypical memory center of the brain, mostly because it is difficult to visualize dendritic spines in this deeply embedded structure with sufficient spatial resolution.

Gouy phase shift measurement using interferometric second-harmonic generation.

We report on a simple way to directly measure the Gouy phase shift of a strongly focused laser beam. This is accomplished by using a recent technique, namely, interferometric second-harmonic generation. We expect that this method will be of interest in a wide range of research fields, from high-harmonic and attosecond pulse generation to femtochemistry and nonlinear microscopy.

In tendons, differing physiological requirements lead to functionally distinct nanostructures.

The collagen-based tissues of animals are hierarchical structures: even tendon, the simplest collagenous tissue, has seven to eight levels of hierarchy. Tailoring tissue structure to match physiological function can occur at many different levels. We wanted to know if the control of tissue architecture to achieve function extends down to the nanoscale level of the individual, cable-like collagen fibrils.

How aging impacts skin biomechanics: a multiscale study in mice.

Skin aging is a complex process that strongly affects the mechanical behavior of skin. This study aims at deciphering the relationship between age-related changes in dermis mechanical behavior and the underlying changes in dermis microstructure. To that end, we use multiphoton microscopy to monitor the reorganization of dermal collagen during mechanical traction assays in ex vivo skin from young and old mice.

A novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization.

Unlabelled: Skin is a complex, multi-layered organ, with important functions in the protection of the body. The dermis provides structural support to the epidermal barrier, and thus has attracted a large number of mechanical studies. As the dermis is made of a mixture of stiff fibres embedded in a soft non-fibrillar matrix, it is classically considered that its mechanical response is based on an initial alignment of the fibres, followed by the stretching of the aligned fibres.

Femoral epiphyseal cartilage matrix changes at predilection sites of equine osteochondrosis: Quantitative MRI, second-harmonic microscopy, and histological findings.

Osteochondrosis is an ischemic chondronecrosis of epiphyseal growth cartilage that results in focal failure of endochondral ossification and osteochondritis dissecans at specific sites in the epiphyses of humans and animals, including horses. The upstream events leading to the focal ischemia remain unknown. The epiphyseal growth cartilage matrix is composed of proteoglycan and collagen macromolecules and encases its vascular tree in canals.

Nonlinear optical properties of calcium barium niobate epitaxial thin films.

We investigate the potential of epitaxial calcium barium niobate (CBN) thin film grown by pulsed laser deposition for optical frequency conversion. Using second harmonic generation (SHG), we analyze the polarization response of the generated signal to determine the ratios d / d and d / d of the three independent components of the second-order nonlinear susceptibility tensor in CBN thin film. In addition, a detailed comparison to the signal intensity obtained in a y-cut quartz allows us to measure the absolute value of these components in CBN thin film: d = 5 ± 2 pm / V, d = 3.

Fast interferometric second harmonic generation microscopy.

We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample.

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy.

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue.

Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy.

Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility.

Analysis of forward and backward Second Harmonic Generation images to probe the nanoscale structure of collagen within bone and cartilage.

Collagen ultrastructure plays a central role in the function of a wide range of connective tissues. Studying collagen structure at the microscopic scale is therefore of considerable interest to understand the mechanisms of tissue pathologies. Here, we use second harmonic generation microscopy to characterize collagen structure within bone and articular cartilage in human knees.