Sindy K. Y. Tang

values. Activation levels of basophils from anonymous and presumed non-allergic donors in samples stored in the lyse/fix buffer for up to 7 days at 4 °C are similar to those analyzed on day 0. In summary, we demonstrate the potential of μF-prep to facilitate access to the BAT by simplifying sample preparation, stabilizing samples to remove the need for overnight blood shipping for flow cytometry analysis, and automating the data analysis pipeline.

Cutting soft materials on the microscale has emerging applications in single-cell studies, tissue microdissection for organoid culture, drug screens, and other analyses. However, the cutting process is complex and remains incompletely understood. Furthermore, precise control over blade geometries, such as the blade tip radius, has been difficult to achieve. In this work, we use the Nanoscribe 3D printer to precisely fabricate micro-blades (i.e., blades <1 mm in length) and blade grid geometries. This fabrication method enables a systematic study of the effect of blade geometry on the indentation cutting of paraffin wax, a common tissue-embedding material. First, we print straight micro-blades with tip radius ranging from ~100 nm to 10 μm. The micro-blades are mounted in a custom nanoindentation setup to measure the cutting energy during indentation cutting of paraffin. Cutting energy, measured as the difference in dissipated energy between the first and second loading cycles, decreases as blade tip radius decreases, until ~357 nm when the cutting energy plateaus despite further decrease in tip radius. Second, we expand our method to blades printed in unconventional configurations, including parallel blade structures and blades arranged in a square grid. Under the conditions tested, the cutting energy scales approximately linearly with the total length of the blades comprising the blade structure. The experimental platform described can be extended to investigate other blade geometries and guide the design of microscale cutting of soft materials.

Cutting soft materials on the microscale has emerging applications in single-cell studies, tissue microdissection for organoid culture, drug screens, and other analyses. However, the cutting process is complex and remains incompletely understood. Furthermore, precise control over blade geometries, such as the blade tip radius, has been difficult to achieve. In this work, we use the Nanoscribe 3D printer to precisely fabricate micro-blades (i.e., blades &lt;1 mm in length) and blade grid geometries. This fabrication method enables a systematic study of the effect of blade geometry on the indentation cutting of paraffin wax, a common tissue-embedding material. First, we print straight micro-blades with tip radius ranging from ~100 nm to 10 um. The micro-blades are mounted in a custom nanoindentation setup to measure the cutting energy during indentation cutting of paraffin. Cutting energy, measured as the difference in dissipated energy between the first and second loading cycles, decreases as blade tip radius decreases, until ~357 nm when the cutting energy plateaus despite further decrease in tip radius. Second, we expand our method to blades printed in unconventional configurations, including parallel blade structures and blades arranged in a square grid. Under the conditions tested, the cutting energy scales approximately linearly with the total length of the blades comprising the blade structure. The experimental platform described can be extended to investigate other blade geometries and guide the design of microscale cutting of soft materials.

The ability to regenerate after the loss of a part is a hallmark of living systems and occurs at both the tissue and organ scales, but also within individual cells. Regeneration entails many processes that are physical and mechanical in nature, including the closure of wounds, the repositioning of material from one place to another, and the restoration of symmetry following perturbations. However, we currently know far more about the genetics and molecular signaling pathways involved in regeneration, and there is a need to investigate the role of physical forces in the process. Here, we will provide an overview of how physical forces may play a role in wound healing and regeneration, in which we compare and contrast regenerative processes at the tissue and cell scales.

Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.

ABSTRACT Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery of Stentor coeruleus (SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery on Stentor as our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 hours without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.