Raman microspectroscopy (RMS) was used to detect and image molecular markers specific to cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs). This technique is noninvasive and thus can be used to discriminate individual live CMs within highly heterogeneous cell populations. Principal component analysis (PCA) of the Raman spectra was used to build a classification model for identification of individual CMs. Retrospective immunostaining imaging was used as the gold standard for phenotypic identification of each cell. We were able to discriminate CMs from other phenotypes with >97% specificity and >96% sensitivity, as calculated with the use of cross-validation algorithms (target 100% specificity). A comparison between Raman spectral images corresponding to selected Raman bands identified by the PCA model and immunostaining of the same cells allowed assignment of the Raman spectral markers. We conclude that glycogen is responsible for the discrimination of CMs, whereas myofibril proteins have a lesser contribution. This study demonstrates the potential of RMS for allowing the noninvasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes, and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro.
Shear fractures can facilitate fluid conductivity through rock. Aperture and roughness are controlling characteristics for a fracture's fluid conductivity. Inspired by en echelon fractures, we develop a shear "fracturelet" model that predicts anisotropic aperture with respect to the direction of shearing, rougher (nonplanar) rather than smoother (planar) fractures, and the bounds of this roughness for a coalesced fracture. This tendency for rougher fracture creation is validated by in situ X-ray images and fluid conductivity measurements from triaxial direct shear experiments on anhydrite and shale. These experiments were conducted at confining stresses from 4 to 30 MPa and shear displacement magnitudes from 0 to 2 mm on initially intact rock specimens. Hydraulic, dilatational, and local fracture apertures were measured in the experiments. Apertures exhibited strong anisotropy with more conductive flow paths forming perpendicular to the direction of shearing. Local and dilatational aperture were found to be positively correlated with increasing shear displacement but hydraulic aperture was found to vary significantly, always having values smaller than the other aperture measures at factors ranging from 0.6 to 0.0. An implication of these results is that shear fractures have a mechanism for simultaneously exhibiting very low fluid conductivity and high fluid storage volume.
Target subsurface reservoirs for emerging low-carbon energy technologies and geologic carbon sequestration typically have low permeability and thus rely heavily on fluid transport through natural and induced fracture networks. Sustainable development of these systems requires deeper understanding of how geochemically mediated deformation impacts fracture microstructure and permeability evolution, particularly with respect to geochemical reactions between pore fluids and the host rock. In this work, a series of triaxial direct shear experiments was designed to evaluate how fractures generated at subsurface conditions respond to penetration of reactive fluids with a focus on the role of mineral precipitation. Calcite-rich shale cores were directly sheared under 3.5 MPa confining pressure using BaCl 2-rich solutions as a working fluid. Experiments were conducted within an X-ray computed tomography (xCT) scanner to capture 4-D evolution of fracture geometry and precipitate growth. Three shear tests evidenced nonuniform precipitation of barium carbonates (BaCO 3) along through-going fractures, where the extent of precipitation increased with increasing calcite content. Precipitates were strongly localized within fracture networks due to mineral, geochemical, and structural heterogeneities and generally concentrated in smaller apertures where rock:water ratios were highest. The combination of elevated fluid saturation and reactive surface area created in freshly activated fractures drove near-immediate mineral precipitation that led to an 80% permeability reduction and significant flow obstruction in the most reactive core. While most previous studies have focused on mixing-induced precipitation, this work demonstrates that fluid-rock interactions can trigger precipitation-induced permeability alterations that can initiate or mitigate risks associated with subsurface energy systems.
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