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Lung diseases such as cancer substantially alter the mechanical properties of the organ with direct impact on the development, progression, diagnosis, and treatment response of diseases. Despite significant interest in the lung’s material properties, measuring the stiffness of intact lungs at sub-alveolar resolution has not been possible. Recently, we developed the crystal ribcage to image functioning lungs at optical resolution while controlling physiological parameters such as air pressure. Here, we introduce a data-driven, multiscale network model that takes images of the lung at different distending pressures, acquired via the crystal ribcage, and produces corresponding absolute stiffness maps. Following validation, we report absolute stiffness maps of the functioning lung at microscale resolution in health and disease. For representative images of a healthy lung and a lung with primary cancer, we find that while the lung exhibits significant stiffness heterogeneity at the microscale, primary tumors introduce even greater heterogeneity into the lung’s microenvironment. Additionally, we observe that while the healthy alveoli exhibit strain-stiffening of ∼1.75 times, the tumor’s stiffness increases by a factor of six across the range of measured transpulmonary pressures. While the tumor stiffness is 1.4 times the lung stiffness at a transpulmonary pressure of three cmH₂O, the tumor’s mean stiffness is nearly five times greater than that of the surrounding tissue at a transpulmonary pressure of 18 cmH₂O. Finally, we report that the variance in both strain and stiffness increases with transpulmonary pressure in both the healthy and cancerous lungs. Our new method allows quantitative assessment of disease-induced stiffness changes in the alveoli with implications for mechanotransduction. 

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real time. Despite advances in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that allows multiscale optical imaging of the functioning lung from whole-organ to single-cell level. It enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal and optogenetic interventions, and it preserves the three-dimensional architecture, air-liquid interface, cellular diversity and respiratory-circulatory functions of the lung. Utilizing these capabilities on murine models of pulmonary pathologies we probed remodeling of respiratory-circulatory functions at the single-alveolus and capillary levels during disease progression. The crystal ribcage and its broad applications presented here will facilitate further studies of nearly any pulmonary disease as well as lead to the identification of new targets for treatment strategies. 

The cytoskeleton, a complex network of protein filaments and crosslinking proteins, dictates diverse cellular processes ranging from division to cargo transport. Yet, the role the cytoskeleton plays in the intracellular transport of DNA and other macromolecules remains poorly understood. Here, using single-molecule conformational tracking, we measure the transport and conformational dynamics of linear and relaxed circular (ring) DNA in composite networks of actin and microtubules with variable types of crosslinking. While both linear and ring DNA undergo anomalous, non-Gaussian, and non-ergodic subdiffusion, the detailed dynamics are controlled by both DNA topology (linear vs. ring) and crosslinking motif. Ring DNA swells, exhibiting heterogeneous subdiffusion controlled via threading by cytoskeleton filaments, while linear DNA compacts, exhibiting transport via caging and hopping. Importantly, while the crosslinking motif has little effect on ring DNA, linear DNA in networks with actin–microtubule crosslinking is significantly less ergodic and shows more heterogeneous transport than with actin–actin or microtubule–microtubule crosslinking. 

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.