Laser Micromachining of Bone as a Tool for Studying Bone Marrow Biology.

The bone marrow (BM) has traditionally been a difficult tissue to access because it is embedded deep within the bone matrix. It is home to the hematopoietic stem cells (HSCs) that give rise to all blood cells in the body. It is also the site of origin for malignant blood cells such as leukemia and multiple myeloma, as well as a frequent site of metastasis for many solid tumors including prostate and breast cancer. The following chapter describes how laser micromachining of bone can be used to improve both optical and physical access to the BM. For example, laser thinning of the overlying bone can improve optical access, enabling deeper imaging into the BM as well as enhancing optical resolution by reducing scattering and aberration. Laser micromachining can also be used to provide physical access into the BM by creating access ports for micropipette insertion and delivery of cells to precise locations in the BM, as well as for the extraction of BM cells and interstitial fluid, all under image guidance. This chapter provides a detailed protocol for installing a laser-micromachining capability for users with an existing multiphoton microscope. Additionally, we briefly outline how such a system improves the optical resolution during imaging as well as its potential use to study injury response.

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.

Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis.

Interactions between the immune and central nervous systems strongly influence brain health. Although the blood-brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain's interstitial and perivascular spaces, facilitates outward signaling beyond the blood-brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull's hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.

From coffee stains to uniform deposits: Significance of the contact-line mobility.

HYPOTHESIS: Contact-line motion upon drying of a sessile droplet strongly affects the solute transport and solvent evaporation profile. Hence, it should have a strong impact on the deposit formation and might be responsible for volcano-like, dome-like and flat deposit morphologies. EXPERIMENTS: A method based on a thin-film interference was used to track the drop height profile and contact line motion during the drying. A diverse set of drying scenarios was obtained by using inks with different solvent compositions and by adjusting the substrate wetting properties. The experimental data was compared to the predictions of a phenomenological model. FINDINGS: We highlight the essential role of contact-line mobility on the deposit morphology of solution-based inks. A pinned contact line produces exclusively ring-like deposits under normal conditions. On the contrary, drops with a mobile contact line can produce ring-, flat- or dome-like morphology. The developed phenomenological model shows that the deposit morphology depends on solvent evaporation profile, evolution of the drop radius relative to its contact angle, and the ratio between initial and maximal (gelling) solute concentration. These parameters can be adjusted by the ink solvent composition and substrate wetting behaviour, which provides a way for deposition of uniform and flat deposits via inkjet printing.

Relative retinal flow velocity detection using optical coherence tomography angiography imaging.

Optical coherence tomography angiography (OCTA) imaging is a valuable tool for the visualization of retinal vasculature at an unprecedented level of details. However, due to relatively long time-interval between repeated scans in the conventional OCTA scanning protocol, the OCTA flow signal suffers from low dynamic range and loss of velocity-intensity correlation. The ability to distinguish fast and slow flow in the retina may provide a powerful tool for the assessment of early-stage retinal diseases such as vein occlusion. Here, we report a method to detect relative flow velocity in human retina using a 67.5 kHz spectral-domain OCTA device. By adapting the selection of A-scan time-intervals within a single OCTA acquisition and combining the resulting OCTA images, we expand the detectable velocity range. After a quantitative validation of this method performing microchannel flow experiments with varying flow velocities, we demonstrate this approach on human eyes using CIRRUS HD-OCT 5000 with AngioPlex (ZEISS, Dublin, CA) through a prototype scanning pattern.

Optical-resonance-assisted generation of super monodisperse microdroplets and microbeads with nanometer precision.

Droplets with predefined sizes have been controllably produced at the tip of a micro-capillary immersed in an external fluid while tracking the high Q-factor whispering gallery modes (WGM). The modes were fitted to a model to give precise real-time size measurement, which was used as a feedback to control the pressure in the capillary and the release of the droplet from the capillary when it reached the target size. In this way a dispersion of highly monodisperse droplets anywhere in the size range from 5 µm to 50 µm were produced. To fabricate solid beads, the droplets were made from a liquid photopolymer and were later polymerized with UV light. The polymerized beads showed long term stability. The diameter of the generated oil droplets and polymerized microbeads could be reproduced with a standard deviation of 1.1 nm and 20 nm, respectively. Overall, the demonstrated method improves the size precision by three and two orders of magnitude for microdroplets and microbeads, respectively, compared to standard production methods such as reported in microfluidics. Encoding of short words and numbers has been demonstrated by producing three beads with predefined sizes. The stored information has been read from the emitted spectrum.

Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy.

In this work, we quantify the mechanical properties of the extra-cellular matrix (ECM) in live zebrafish using Brillouin microscopy. Optimization of the imaging conditions and parameters, combined with careful spectral analysis, allows us to resolve the thin ECM and distinguish its Brillouin frequency shift, a proxy for mechanical properties, from the surrounding tissue. High-resolution mechanical mapping further enables the direct measurement of the thickness of the ECM label-free and in-vivo. We find the ECM to be ~500 nm thick, and in very good agreement with electron microscopy quantification. Our results open the door for future studies that aim to investigate the role of ECM mechanics for zebrafish morphogenesis and axis elongation.