Near-lifespan longitudinal tracking of brain microvascular morphology, topology, and flow in male mice.

In age-related neurodegenerative diseases, pathology often develops slowly across the lifespan. As one example, in diseases such as Alzheimer's, vascular decline is believed to onset decades ahead of symptomology. However, challenges inherent in current microscopic methods make longitudinal tracking of such vascular decline difficult. Here, we describe a suite of methods for measuring brain vascular dynamics and anatomy in mice for over seven months in the same field of view. This approach is enabled by advances in optical coherence tomography (OCT) and image processing algorithms including deep learning. These integrated methods enabled us to simultaneously monitor distinct vascular properties spanning morphology, topology, and function of the microvasculature across all scales: large pial vessels, penetrating cortical vessels, and capillaries. We have demonstrated this technical capability in wild-type and 3xTg male mice. The capability will allow comprehensive and longitudinal study of a broad range of progressive vascular diseases, and normal aging, in key model systems.

Targeted capillary photothrombosis via multiphoton excitation of Rose Bengal.

Microvascular stalling, the process occurring when a capillary temporarily loses perfusion, has gained increasing interest in recent years through its demonstrated presence in various neuropathologies. Studying the impact of such stalls on the surrounding brain tissue is of paramount importance to understand their role in such diseases. Despite efforts trying to study the stalling events, investigations are hampered by their elusiveness and scarcity. In an attempt to alleviate these hurdles, we present here a novel methodology enabling transient occlusions of targeted microvascular segments through multiphoton excitation of Rose Bengal, an established photothrombotic agent. With n = 7 mice C57BL/6 J (5 males and 2 females) and 95 photothrombosis trials, we demonstrate the ability of triggering reversible blockages by illuminating a capillary segment during ∼300 s at 1000 nm, using a standard Ti:Sapphire femtosecond laser. Furthermore, we performed concurrent Optical Coherence Microscopy (OCM) angiography imaging of the microvascular network to highlight the specificity of the targeted occlusion and its duration. Through comparison with a control group, we conclude that blood flow cessation is indeed created by the photothrombotic agent via multiphoton excitation and is temporary, followed by a flow recovery in less than 24 h. Moreover, Immunohistology points toward a stalling mechanism driven by adherence of the neutrophil in the vascular lumen. This observation seems to be promoted by the inflammation locally created via multiphoton activation of Rose Bengal.

Digital aberration correction enhances field of view in visible-light optical coherence microscopy.

In optical coherence microscopy, optical aberrations commonly result in astigmatism-dominated wavefront errors in the peripheral regions of the optical objective, primarily elongating the microscope's point-spread function along the radial direction in the vicinity of the focal plane. We report on enhanced-field-of-view optical coherence microscopy through computational aberration correction in the visible-light range. An isotropic spatial resolution of 2.5 µm was achieved over an enhanced lateral field of view spanning 1.3 mm × 1.6 mm, as experimentally verified in a micro-bead phantom and further demonstrated in ex vivo tissue samples. The extended field of view achieved by the digital aberration correction facilitates the use of low-cost systems by averting the need for high-quality objectives.

Agronomic characterization of anaerobic digestates with near-infrared spectroscopy.

Anaerobic digestion is an increasingly widespread process for organic waste treatment and renewable energy production due to the methane content of the biogas. This biological process also produces a digestate (i.e., the remaining content of the waste after treatment) with a high fertilizing potential. The digestate composition is highly variable due to the various organic wastes used as feedstock, the different plant configurations, and the post-treatment processes used. In order to optimize digestate spreading on agricultural soils by optimizing the fertilizer dose and, thus, reducing environmental impacts associated to digestate application, the agronomic characterization of digestate is essential. This study investigates the use of near infrared spectroscopy for predicting the most important agronomic parameters from freeze-dried digestates. A data set of 193 digestates was created to calibrate partial least squares regression models predicting organic matter, total organic carbon, organic nitrogen, phosphorus, and potassium contents. The calibration range of the models were between 249.8 and 878.6 gOM.kgDM-1, 171.9 and 499.5 gC.kgDM-1, 5.3 and 74.1 gN.kgDM-1, 2.7 and 44.9 gP.kgDM-1 and between 0.5 and 171.8 gK.kgDM-1, respectively. The calibrated models reliably predicted organic matter, total organic carbon, and phosphorus contents for the whole diversity of digestates with root mean square errors of prediction of 70.51 gOM.kgDM-1, 34.84 gC.kgDM-1 and 4.08 gP.kgDM-1, respectively. On the other hand, the model prediction of the organic nitrogen content had a root mean square error of 7.55 gN.kgDM-1 and was considered as acceptable. Lastly, the results did not demonstrate the feasibility of predicting the potassium content in digestates with near infrared spectroscopy. These results show that near infrared spectroscopy is a very promising analytical method for the characterization of the fertilizing value of digestates, which could provide large benefits in terms of analysis time and cost.

Deep Learning and Simulation for the Estimation of Red Blood Cell Flux With Optical Coherence Tomography.

We present a deep learning and simulation-based method to measure cortical capillary red blood cell (RBC) flux using Optical Coherence Tomography (OCT). This method is more accurate than the traditional peak-counting method and avoids any user parametrization, such as a threshold choice. We used data that was simultaneously acquired using OCT and two-photon microscopy to uncover the distribution of parameters governing the height, width, and inter-peak time of peaks in OCT intensity associated with the passage of RBCs. This allowed us to simulate thousands of time-series examples for different flux values and signal-to-noise ratios, which we then used to train a 1D convolutional neural network (CNN). The trained CNN enabled robust measurement of RBC flux across the entire network of hundreds of capillaries.

A simulation study investigating potential diffusion-based MRI signatures of microstrokes.

Recent studies suggested that cerebrovascular micro-occlusions, i.e. microstokes, could lead to ischemic tissue infarctions and cognitive deficits. Due to their small size, identifying measurable biomarkers of these microvascular lesions remains a major challenge. This work aims to simulate potential MRI signatures combining arterial spin labeling (ASL) and multi-directional diffusion-weighted imaging (DWI). Driving our hypothesis are recent observations demonstrating a radial reorientation of microvasculature around the micro-infarction locus during recovery in mice. Synthetic capillary beds, randomly- and radially-oriented, and optical coherence tomography (OCT) angiograms, acquired in the barrel cortex of mice (n = 5) before and after inducing targeted photothrombosis, were analyzed. Computational vascular graphs combined with a 3D Monte-Carlo simulator were used to characterize the magnetic resonance (MR) response, encompassing the effects of magnetic field perturbations caused by deoxyhemoglobin, and the advection and diffusion of the nuclear spins. We quantified the minimal intravoxel signal loss ratio when applying multiple gradient directions, at varying sequence parameters with and without ASL. With ASL, our results demonstrate a significant difference (p < 0.05) between the signal-ratios computed at baseline and 3 weeks after photothrombosis. The statistical power further increased (p < 0.005) using angiograms measured at week 4. Without ASL, no reliable signal change was found. We found that higher ratios, and accordingly improved significance, were achieved at lower magnetic field strengths (e.g., B0 = 3T) and shorter echo time TE (< 16 ms). Our simulations suggest that microstrokes might be characterized through ASL-DWI sequence, providing necessary insights for posterior experimental validations, and ultimately, future translational trials.

Soliton microcomb based spectral domain optical coherence tomography.

Spectral domain optical coherence tomography (OCT) is a widely employed, minimally invasive bio-medical imaging technique, which requires a broadband light source, typically implemented by super-luminescent diodes. Recent advances in soliton based photonic integrated frequency combs (soliton microcombs) have enabled the development of low-noise, broadband chipscale frequency comb sources, whose potential for OCT imaging has not yet been unexplored. Here, we explore the use of dissipative Kerr soliton microcombs in spectral domain OCT and show that, by using photonic chipscale Si3N4 resonators in conjunction with 1300 nm pump lasers, spectral bandwidths exceeding those of commercial OCT sources are possible. We characterized the exceptional noise properties of our source (in comparison to conventional OCT sources) and demonstrate that the soliton states in microresonators exhibit a residual intensity noise floor at high offset frequencies that is ca. 3 dB lower than a traditional OCT source at identical power, and can exhibit significantly lower noise performance for powers at the milli-Watt level. Moreover, we demonstrate that classical amplitude noise of all soliton comb teeth are correlated, i.e., common mode, in contrast to superluminescent diodes or incoherent microcomb states, which opens a new avenue to improve imaging speed and performance beyond the thermal noise limit.

Validation of red blood cell flux and velocity estimations based on optical coherence tomography intensity fluctuations.

We present a validation of red blood cell flux and speed measurements based on the passage of erythrocytes through the OCT's focal volume. We compare the performance of the so-called RBC-passage OCT technique to co-localized and simultaneously acquired two-photon excitation fluorescence microscopy (TPEF) measurements. Using concurrent multi-modal imaging, we show that fluctuations in the OCT signal display highly similar features to TPEF time traces. Furthermore, we demonstrate an overall difference in RBC flux and speed of 2.5 ± 3.27 RBC/s and 0.12 ± 0.67 mm/s (mean ± S.D.), compared to TPEF. The analysis also revealed that the OCT RBC flux estimation is most accurate between 20 RBC/s to 60 RBC/s, and is severely underestimated at fluxes beyond 80 RBC/s. Lastly, our analysis shows that the RBC speed estimations increase in accuracy as the speed decreases, reaching a difference of 0.16 ± 0.25 mm/s within the 0-0.5 mm/s speed range.

Automated Analysis of Brain Microvasculature: From Segmentation to Anatomical Modeling.

Two-photon microscopy (TPM) can provide a detailed microscopic information of cerebrovascular structures. Extracting anatomical vascular models from TPM angiograms remains a tedious task due to image degeneration associated with TPM acquisitions and the complexity of microvascular networks. Here, we propose a fully automated pipeline capable of providing useful anatomical models of vascular structures captured with TPM. In the proposed method, we segment blood vessels using a fully convolutional neural network and employ the resulting binary labels to create an initial geometric graph enclosed within vessels boundaries. The initial geometry is then decimated and refined to form graphed curve skeletons that can retain both the vascular shape and its topology. We validate the proposed method on 3D realistic TPM angiographies and compare our results with that obtained through manual annotations.

Longitudinal optical coherence tomography imaging of tissue repair and microvasculature regeneration and function after targeted cerebral ischemia.

SIGNIFICANCE: Understanding how the brain recovers from cerebral tissue and vascular damage after an ischemic event can help develop new therapeutic strategies for the treatment of stroke. AIM: We investigated cerebral tissue repair and microvasculature regeneration and function after a targeted ischemic stroke. APPROACH: Following photothrombosis occlusion of microvasculature, chronic optical coherence tomography (OCT)-based angiography was used to track ischemic tissue repair and microvasculature regeneration at three different cortical depths and up to 28 days in awake animals. Capillary network orientation analysis was performed to study the structural pattern of newly formed microvasculature. Based on the time-resolved OCT-angiography, we also investigated capillary stalling, which is likely related to ischemic stroke-induced inflammation. RESULTS: Deeper cerebral tissue was found to have a larger ischemic area than shallower regions at any time point during the course of poststroke recovery, which suggests that cerebral tissue located deep in the cortex is more vulnerable. Regenerated microvasculature had a highly organized pattern at all cortical depths with a higher degree of structural reorganization in deeper regions. Additionally, capillary stalling event analysis revealed that cerebral ischemia augmented stalling events considerably. CONCLUSION: Longitudinal OCT angiography reveals that regenerated capillary network has a highly directional pattern and an increased density and incidence of capillary stalling event.

ATR-FTIR Microspectroscopy Brings a Novel Insight Into the Study of Cell Wall Chemistry at the Cellular Level.

Wood is a complex tissue that fulfills three major functions in trees: water conduction, mechanical support and nutrient storage. In Angiosperm trees, vessels, fibers and parenchyma rays are respectively assigned to these functions. Cell wall composition and structure strongly varies according to cell type, developmental stages and environmental conditions. This complexity can therefore hinder the study of the molecular mechanisms of wood formation, underlying the construction of its properties. However, this can be circumvented thanks to the development of cell-specific approaches and microphenotyping. Here, we present a non-destructive microphenotyping method based on attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) microspectroscopy. We applied this technique to three types of poplar wood: normal wood of staked trees (NW), tension and opposite wood of artificially tilted trees (TW, OW). TW is produced by angiosperm trees in response to mechanical strains and is characterized by the presence of G fibers, exhibiting a thick gelatinous extra-layer, named G-layer, located in place of the usual S2 and/or S3 layers. By contrast, OW located on the opposite side of the trunk is totally deprived of fibers with G-layers. We developed a workflow for hyperspectral image analysis with both automatic pixel clustering according to cell wall types and identification of differentially absorbed wavenumbers (DAWNs). As pixel clustering failed to assign pixels to ray S-layers with sufficient efficiency, the IR profiling and identification of DAWNs were restricted to fiber and vessel cell walls. As reported elsewhere, this workflow identified cellulose as the main component of the G-layers, while the amount in acetylated xylans and lignins were shown to be reduced. These results validate ATR-FTIR technique for in situ characterization of G layers. In addition, this study brought new information about IR profiling of S-layers in TW, OW and NW. While OW and NW exhibited similar profiles, TW fibers S-layers combined characteristics of TW G-layers and of regular fiber S-layers. Unexpectedly, vessel S-layers of the three kinds of wood showed significant differences in IR profiling. In conclusion, ATR-FTIR microspectroscopy offers new possibilities for studying cell wall composition at the cell level.

Supervised learning to quantify amyloidosis in whole brains of an Alzheimer's disease mouse model acquired with optical projection tomography.

Alzheimer's disease (AD) is characterized by amyloidosis of brain tissues. This phenomenon is studied with genetically-modified mouse models. We propose a method to quantify amyloidosis in whole 5xFAD mouse brains, a model of AD. We use optical projection tomography (OPT) and a random forest voxel classifier to segment and measure amyloid plaques. We validate our method in a preliminary cross-sectional study, where we measure 6136 ± 1637, 8477 ± 3438, and 17267 ± 4241 plaques (AVG ± SD) at 11, 17, and 31 weeks. Overall, this method can be used in the evaluation of new treatments against AD.

Optical alignment device for two-photon microscopy.

Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here, we first expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover slide) and compare these results with an objective devoid of collar-correction. Second, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window to an angle below 0.25°, thereby significantly improving the imaging quality. Finally, we describe a tilt-correction procedure for users in an in vivo setting, enabling the accurate alignment with a resolution of <0.2° in only few iterations.

Imaging of cortical structures and microvasculature using extended-focus optical coherence tomography at 1.3 µm.

Extended-focus optical coherence tomography (xf-OCT) is a variant of optical coherence tomography (OCT) wherein the illumination and/or detection modes are engineered to provide a constant diffractionless lateral resolution over an extended depth of field (typically 3 to 10× the Rayleigh range). xf-OCT systems operating at 800 nm have been devised and used in the past to image brain structures at high-resolution in vivo, but are limited to ∼500 µm in penetration depth due to their short illumination wavelength. Here we present an xf-OCT system optimized to an image deeper within the cortex by using a longer illumination central wavelength of 1310 nm. The system offers a lateral resolution of 3 and 6.5 µm, over a depth of 900 µm and >1.5 mm using a 10× and 5× objective, respectively, in air. We characterize the system's resolution using microbeads embedded in PDMS and demonstrate its capabilities by imaging the cortical structure and microvasculature in anesthetized mice to a depth of ∼0.8 mm. Finally, we illustrate the difference in penetration depths obtainable with the new system and an xf-OCT system operating at 800 nm.

In vivo high-resolution cortical imaging with extended-focus optical coherence microscopy in the visible-NIR wavelength range.

Visible light optical coherence tomography has shown great interest in recent years for spectroscopic and high-resolution retinal and cerebral imaging. Here, we present an extended-focus optical coherence microscopy system operating from the visible to the near-infrared wavelength range for high axial and lateral resolution imaging of cortical structures in vivo. The system exploits an ultrabroad illumination spectrum centered in the visible wavelength range (λc = 650 nm, Δλ ∼ 250 nm) offering a submicron axial resolution (∼0.85 µm in water) and an extended-focus configuration providing a high lateral resolution of ∼1.4 µm maintained over ∼150 µm in depth in water. The system's axial and lateral resolution are first characterized using phantoms, and its imaging performance is then demonstrated by imaging the vasculature, myelinated axons, and neuronal cells in the first layers of the somatosensory cortex of mice in vivo.

Interferometric synthetic aperture microscopy for extended focus optical coherence microscopy.

Optical coherence microscopy (OCM) is an interferometric technique providing 3D images of biological samples with micrometric resolution and penetration depth of several hundreds of micrometers. OCM differs from optical coherence tomography (OCT) in that it uses a high numerical aperture (NA) objective to achieve high lateral resolution. However, the high NA also reduces the depth-of-field (DOF), scaling with 1/NA2. Interferometric synthetic aperture microscopy (ISAM) is a computed imaging technique providing a solution to this trade-off between resolution and DOF. An alternative hardware method to achieve an extended DOF is to use a non-Gaussian illumination. Extended focus OCM (xfOCM) uses a Bessel beam to obtain a narrow and extended illumination volume. xfOCM detects back-scattered light using a Gaussian mode in order to maintain good sensitivity. However, the Gaussian detection mode limits the DOF. In this work, we present extended ISAM (xISAM), a method combining the benefits of both ISAM and xfOCM. xISAM uses the 3D coherent transfer function (CTF) to generalize the ISAM algorithm to different system configurations. We demonstrate xISAM both on simulated and experimental data, showing that xISAM attains a combination of high transverse resolution and extended DOF which has so far been unobtainable through conventional ISAM or xfOCM individually.

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography.

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 µm axial and 0.4 µm lateral resolution maintained over a depth of 40 µm, while preserving the advantages of Fourier domain OCM. Our system uses an ultra-broad spectrum from a supercontinuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the system visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo brain tissue of both healthy and alzheimeric mice. In addition to neuronal cell bodies, fibers and plaques, visOCM imaging of brain tissue reveals fine vascular structures and sub-cellular features through its high spatial resolution. Sub-cellular structures were also observed in live cells and were further revealed through a protocol traditionally used for OCT angiography.

Label-free three-dimensional imaging of Caenorhabditis elegans with visible optical coherence microscopy.

Fast, label-free, high-resolution, three-dimensional imaging platforms are crucial for high-throughput in vivo time-lapse studies of the anatomy of Caenorhabditis elegans, one of the most commonly used model organisms in biomedical research. Despite the needs, methods combining all these characteristics have been lacking. Here, we present label-free imaging of live Caenorhabditis elegans with three-dimensional sub-micrometer resolution using visible optical coherence microscopy (visOCM). visOCM is a versatile optical imaging method which we introduced recently for tomography of cell cultures and tissue samples. Our method is based on Fourier domain optical coherence tomography, an interferometric technique that provides three-dimensional images with high sensitivity, high acquisition rate and micrometer-scale resolution. By operating in the visible wavelength range and using a high NA objective, visOCM attains lateral and axial resolutions below 1 µm. Additionally, we use a Bessel illumination offering an extended depth of field of approximately 40 µm. We demonstrate that visOCM's imaging properties allow rapid imaging of full sized living Caenorhabditis elegans down to the sub-cellular level. Our system opens the door to many applications such as the study of phenotypic changes related to developmental or ageing processes.

Community gardening in poor neighborhoods in France: A way to re-think food practices?

Social inequalities in diet are attributed to sociocultural determinants, economic constraints, and unequal access to healthy food. Fruits and vegetables are lacking in the diets of disadvantaged populations. The objective was to test the hypothesis that, in poor neighborhoods, community gardeners will have larger supply of healthy food, especially fruit and vegetables, than non-gardeners. We examined community gardens from the perspective of production, economics and nutrition, and social and symbolic dimensions, through multidisciplinary investigations involving women with access to a community garden plot in a poor neighborhood of Marseille, France. Gardeners' monthly household food supplies (purchases and garden production) were analyzed and compared with those of women with a similar socio-economic profile living in the same neighborhoods, without access to a garden. Twenty-one gardeners participated. Only eleven of them harvested during the month of the study, and the amount they collected averaged 53 g of produce per household member per day. Whether they harvested or not, most gardeners gave preference to diversity, taste and healthiness of produce over quantity produced. Interviews revealed a value assigned to social, cultural and symbolic dimensions: pride in producing and cooking their own produce, related self-esteem, and sharing their produce at the meal table. The only significant difference between the food supplies of gardener and non-gardener households was seen for fruit and vegetables (369 vs. 211 g/d per person). This difference was due to larger purchases of fruit and vegetables, and not to higher quantities produced. In spite of the cross-sectional nature of our study and the small quantities harvested, our results suggest that having access to a community garden could encourage socio-economically disadvantaged women to adopt dietary practices that more closely meet dietary recommendations.

3D Time-lapse Imaging and Quantification of Mitochondrial Dynamics.

We present a 3D time-lapse imaging method for monitoring mitochondrial dynamics in living HeLa cells based on photothermal optical coherence microscopy and using novel surface functionalization of gold nanoparticles. The biocompatible protein-based biopolymer coating contains multiple functional groups which impart better cellular uptake and mitochondria targeting efficiency. The high stability of the gold nanoparticles allows continuous imaging over an extended time up to 3000 seconds without significant cell damage. By combining temporal autocorrelation analysis with a classical diffusion model, we quantify mitochondrial dynamics and cast these results into 3D maps showing the heterogeneity of diffusion parameters across the whole cell volume.