Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.

Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging.

Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

Dynamic super-resolution structured illumination imaging in the living brain.

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning.

Rapid autofocusing over long distances is critical for tracking 3D topological variations and sample motion in real time. Taking advantage of a deformable mirror and Shack-Hartmann wavefront sensor, remote focusing can permit fast axial scanning with simultaneous correction of system-induced aberrations. Here, we report an autofocusing technique that combines remote focusing with sequence-dependent learning via a bidirectional long short term memory network. A 120 µm autofocusing range was achieved in a compact reflectance confocal microscope both in air and in refractive-index-mismatched media, with similar performance under arbitrary-thickness liquid layers up to 1 mm. The technique was validated on sample types not used for network training, as well as for tracking of continuous axial motion. These results demonstrate that the proposed technique is suitable for real-time aberration-free autofocusing over a large axial range, and provides unique advantages for biomedical, holographic and other related applications.

Volumetric two-photon fluorescence imaging of live neurons using a multimode optical fiber.

Multimode optical fibers (MMFs), combined with wavefront control methods, have achieved minimally invasive in vivo imaging of neurons in deep-brain regions with diffraction-limited spatial resolution. Here, we report a method for volumetric two-photon fluorescence imaging with a MMF-based system requiring a single transmission matrix measurement. Central to this method is the use of a laser source able to generate both continuous wave light and femtosecond pulses. The chromatic dispersion of pulses generated an axially elongated excitation focus, which we used to demonstrate volumetric imaging of neurons and their dendrites in live rat brain slices through a 60 µm core MMF.

Direct measurement of local oxygen concentration in the bone marrow of live animals.

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Active compensation of extrinsic polarization errors using adaptive optics.

We present a scheme for active compensation of complex extrinsic polarization perturbations introduced into an optical system. Imaging polarimeter is used to measure the polarization state across a beam profile and a liquid crystal spatial light modulator controls the polarization of the input beam. A sequence of measurements permits determination of the birefringence properties of a perturbing specimen. The necessary correction is calculated and fed back to the polarization modulator to compensate for the polarization perturbation. The system capabilities are demonstrated on a range of birefringent specimens.

Focusing light in biological tissue through a multimode optical fiber: refractive index matching.

Controlling light propagation through a step-index multimode optical fiber (MMF) has several important applications, including biological imaging. However, little consideration has been given to the coupling of fiber and tissue optics. In this Letter, we characterized the effects of tissue-induced light distortions, in particular those arising from a mismatch in the refractive index of the pre-imaging calibration and biological media. By performing the calibration in a medium matching the refractive index of the brain, optimal focusing ability was achieved, as well as a gain in focus uniformity within the field-of-view. These changes in illumination resulted in a 30% improvement in spatial resolution and intensity in fluorescence images of beads and live brain tissue. Beyond refractive index matching, our results demonstrate that sample-induced aberrations can severely deteriorate images from MMF-based systems.

Molecular Order of Arterial Collagen Using Circular Polarization Second-Harmonic Generation Imaging.

Second-harmonic generation (SHG) originates from the interaction between upconverted fields from individual scatterers. This renders SHG microscopy highly sensitive to molecular distribution. Here, we aim to take advantage of the difference in SHG between aligned and partially aligned molecules to probe the degree of molecular order during biomechanical testing, independently of the absolute orientation of the scattering molecules. Toward this goal, we implemented a circular polarization SHG imaging approach and used it to quantify the intensity change associated with collagen fibers straightening in the arterial wall during mechanical stretching. We were able to observe the delayed alignment of collagen fibers during mechanical loading, thus demonstrating a simple method to characterize molecular distribution using intensity information alone.

Contribution of collagen fiber undulation to regional biomechanical properties along porcine thoracic aorta.

As major extracellular matrix components, elastin, and collagen play crucial roles in regulating the mechanical properties of the aortic wall and, thus, the normal cardiovascular function. The mechanical properties of aorta, known to vary with age and multitude of diseases as well as the proximity to the heart, have been attributed to the variations in the content and architecture of wall constituents. This study is focused on the role of layer-specific collagen undulation in the variation of mechanical properties along the porcine descending thoracic aorta. Planar biaxial tensile tests are performed to characterize the hyperelastic anisotropic mechanical behavior of tissues dissected from four locations along the thoracic aorta. Multiphoton microscopy is used to image the associated regional microstructure. Exponential-based and recruitment-based constitutive models are used to account for the observed mechanical behavior while considering the aortic wall as a composite of two layers with independent properties. An elevated stiffness is observed in distal regions compared to proximal regions of thoracic aorta, consistent with sharper and earlier collagen recruitment estimated for medial and adventitial layers in the models. Multiphoton images further support our prediction that higher stiffness in distal regions is associated with less undulation in collagen fibers. Recruitment-based models further reveal that regardless of the location, collagen in the media is recruited from the onset of stretching, whereas adventitial collagen starts to engage with a delay. A parameter sensitivity analysis is performed to discriminate between the models in terms of the confidence in the estimated model parameters.

Arterial extracellular matrix: a mechanobiological study of the contributions and interactions of elastin and collagen.

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.

Repeated imaging through a multimode optical fiber using adaptive optics.

Multimode optical fibers (MMF) have shown considerable potential for minimally invasive diffraction-limited fluorescence imaging of deep brain regions owing to their small size. They also look to be suitable for imaging across long time periods, with repeated measurements performed within the same brain region, which is useful to assess the role of synapses in normal brain function and neurological disease. However, the approach is not without challenge. Prior to imaging, light propagation through a MMF must be characterized in a calibration procedure. Manual repositioning, as required for repeated imaging, renders this calibration invalid. In this study, we provide a two-step solution to the problem consisting of (1) a custom headplate enabling precise reinsertion of the MMF implant achieving low-quality focusing and (2) sensorless adaptive optics to correct translational shifts in the MMF position enabling generation of high-quality imaging foci. We show that this approach achieves fluorescence imaging after repeated removal and reinsertion of a MMF.

Adaptive optics for high-resolution imaging.

Adaptive optics (AO) is a technique that corrects for optical aberrations. It was originally proposed to correct for the blurring effect of atmospheric turbulence on images in ground-based telescopes and was instrumental in the work that resulted in the Nobel prize-winning discovery of a supermassive compact object at the centre of our galaxy. When AO is used to correct for the eye's imperfect optics, retinal changes at the cellular level can be detected, allowing us to study the operation of the visual system and to assess ocular health in the microscopic domain. By correcting for sample-induced blur in microscopy, AO has pushed the boundaries of imaging in thick tissue specimens, such as when observing neuronal processes in the brain. In this primer, we focus on the application of AO for high-resolution imaging in astronomy, vision science and microscopy. We begin with an overview of the general principles of AO and its main components, which include methods to measure the aberrations, devices for aberration correction, and how these components are linked in operation. We present results and applications from each field along with reproducibility considerations and limitations. Finally, we discuss future directions.

Deconvolution for multimode fiber imaging: modeling of spatially variant PSF.

Focusing light through a step-index multimode optical fiber (MMF) using wavefront control enables minimally-invasive endoscopy of biological tissue. The point spread function (PSF) of such an imaging system is spatially variant, and this variation limits compensation for blurring using most deconvolution algorithms as they require a uniform PSF. However, modeling the spatially variant PSF into a series of spatially invariant PSFs re-opens the possibility of deconvolution. To achieve this we developed svmPSF: an open-source Java-based framework compatible with ImageJ. The approach takes a series of point response measurements across the field-of-view (FOV) and applies principal component analysis to the measurements' co-variance matrix to generate a PSF model. By combining the svmPSF output with a modified Richardson-Lucy deconvolution algorithm, we were able to deblur and regularize fluorescence images of beads and live neurons acquired with a MMF, and thus effectively increasing the FOV.

Compact and contactless reflectance confocal microscope for neurosurgery.

Visual guidance at the cellular level during neurosurgical procedures is essential for complete tumour resection. We present a compact reflectance confocal microscope with a 20 mm working distance that provided <1.2 µm spatial resolution over a 600 µm × 600 µm field of view in the near-infrared region. A physical footprint of 200 mm × 550 mm was achieved using only standard off-the-shelf components. Theoretical performance of the optical design was first evaluated via commercial Zemax software. Then three specimens from rodents: fixed brain, frozen calvaria and live hippocampal slices, were used to experimentally assess system capability and robustness. Results show great potential for the proposed system to be translated into use as a next generation label-free and contactless neurosurgical microscope.

Fast widefield imaging of neuronal structure and function with optical sectioning in vivo.

Optical microscopy, owing to its noninvasiveness and subcellular resolution, enables in vivo visualization of neuronal structure and function in the physiological context. Optical-sectioning structured illumination microscopy (OS-SIM) is a widefield fluorescence imaging technique that uses structured illumination patterns to encode in-focus structures and optically sections 3D samples. However, its application to in vivo imaging has been limited. In this study, we optimized OS-SIM for in vivo neural imaging. We modified OS-SIM reconstruction algorithms to improve signal-to-noise ratio and correct motion-induced artifacts in live samples. Incorporating an adaptive optics (AO) module to OS-SIM, we found that correcting sample-induced optical aberrations was essential for achieving accurate structural and functional characterizations in vivo. With AO OS-SIM, we demonstrated fast, high-resolution in vivo imaging with optical sectioning for structural imaging of mouse cortical neurons and zebrafish larval motor neurons, and functional imaging of quantal synaptic transmission at Drosophila larval neuromuscular junctions.

Micromechanics of elastic lamellae: unravelling the role of structural inhomogeneity in multi-scale arterial mechanics.

Microstructural deformation of elastic lamellae plays important roles in maintaining arterial tissue homeostasis and regulating vascular smooth muscle cell fate. Our study unravels the underlying microstructural origin that enables elastic lamellar layers to evenly distribute the stresses through the arterial wall caused by intraluminal distending pressure, a fundamental requirement for tissue and cellular function. A new experimental approach was developed to quantify the spatial organization and unfolding of elastic lamellar layers under pressurization in mouse carotid arteries by coupling physiological extension-inflation and multiphoton imaging. Tissue-level circumferential stretch was obtained from analysis of the deformation of a thick-walled cylinder. Our results show that the unfolding and extension of lamellar layers contribute simultaneously to tissue-level deformation. The inner lamellar layers are wavier and unfold more than the outer layers. This waviness gradient compensates the larger tissue circumferential stretch experienced at the inner surface, thus equalizing lamellar layer extension through the arterial wall. Discoveries from this study reveal the importance of structural inhomogeneity in maintaining tissue homeostasis through the arterial wall, and may have profound implications on vascular remodelling in aging and diseases, as well as in tissue engineering of functional blood vessels.

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.