Microscale Measurements of Cell and Tissue Mechanics in Three Dimensions

Two-dimensional (2D) studies have revealed that mechanical forces drive cell migration and can feedback to regulate proliferation, differentiation and the synthesis/remodeling of extracellular matrix (ECM) proteins. Whether these observations can be translated to clinical settings or be utilized for tissue engineering will depend critically on our ability to translate these findings into physiologically relevant three-dimensional (3D) environments. The general goal of this dissertation has been to develop and apply new technologies capable of extending studies of cell and tissue mechanics into 3D environments. In the first project, we measured both shear and normal traction forces exerted by cells cultured on planar substrates. We observed that focal adhesions serve as pivots about which cells generate rotational moments. In the second project, we combined enzymatically degradable synthetic hydrogels with finite element models to measure the mechanical tractions exerted by cells fully encapsulated within 3D matrices. We found that cells reach out thin protrusions and pull back inward towards the cell body with the highest forces at the tip. Cellular extensions that were invading into the surrounding matrix displayed a strong inward force 10-15 microns behind the leading tip, suggesting that growing extensions may establish a contractile waypoint, before invading further. To study the forces cells exert during tissue remodeling, we utilized photolithograpy to generate arrays of microtissues consisting of cells encapsulated in 3D collagen matrices. Microcantilevers were used to constrain the remodeling of the collagen gel and to report the forces generated during this process. We used this technique to explore the effects of boundary stiffness and matrix density within model tendon and cardiac tissues. Finally, we combined this system with a Foerster radius energy transfer (FRET) based biosensor of fibronectin conformation to reveal how tissue geometry and cell-genereated tractions cooperate to pattern matrix conformation during tissue remodeling. Together, these studies highlight novel approaches to understand the nature of cell-ECM interactions in 3D matrices. Such mechanical insights will help us to understand how physical forces drive cell migration and behavior within physiologically relevant environments

Interview with Dale Grassbaugh

Dale Grassbaugh, the father of the Grassbaugh family, talks about his family farm and about how farming has become more of a business now than simply a way of life. The business aspect is strong for the local farmers. He discusses the economic aspects for local farmers in detail.https://digital.kenyon.edu/ffp_interviews/1023/thumbnail.jp

Interview with Auctioneer John \u27Red\u27 Shaw

John Shaw discusses growing up on a farm and being in 4H, and getting involved in the auction business; specifically getting involved with agriculture sales. He also discusses how the new buyers are buying bigger sections of land and have bigger equipment, they are less likely to be small, family farms. He says the biggest change he\u27s noticed in his time is that the farms are becoming bigger and the owners are not young people, because it is so expensive to start a farm.https://digital.kenyon.edu/ffp_interviews/1038/thumbnail.jp

Interview with Dwayne Grassbaugh

Dwayne Grassbaugh discusses going to Virginia Tech and taking care of the dairy section of the farm. He also discusses what a family farm is actually like.https://digital.kenyon.edu/ffp_interviews/1031/thumbnail.jp

Interview with Cindy Grassbaugh

Cindy Grassbaugh, the second daughter of the Grassbaugh\u27s talks of growing up on a farm and having to feed the calves. She also speaks of how her brothers had more responsibilities on the farm because they were physically stronger. Cindy also talks about how she learned responsibility on the farm, and through the 4-H program.https://digital.kenyon.edu/ffp_interviews/1022/thumbnail.jp

Interview with Kathy Grassbaugh

Kathy Grassbaugh, the wife and mother of the Grassbaugh family, discusses life on a farm and whether people are born into farming or whether they can start farming at any age with any background. She also discusses how farms are getting bigger and bigger just to bring in the same prices, because of the agricultural economics at the time. However, she also discusses how being a farmer is like being your own boss and you get to control your income.https://digital.kenyon.edu/ffp_interviews/1007/thumbnail.jp

Interview with Anna Grassbaugh

Anna Grassbaugh discusses growing up on a farm, and how that has changed her outlook on life. Grassbaugh discusses her future goal of working for farmers.https://digital.kenyon.edu/ffp_interviews/1005/thumbnail.jp

Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER "sheet" proliferation after overexpression of tubular junction-forming proteins

Posterior approach to correct for focal plane offsets in lattice light-sheet structured illumination microscopy

Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions

Active PSF shaping and adaptive optics enable volumetric localization microscopy through brain sections

Application of single-molecule switching nanoscopy (SMSN) beyond the coverslip surface poses substantial challenges due to sample-induced aberrations that distort and blur single-molecule emission patterns. We combined active shaping of point spread functions and efficient adaptive optics to enable robust 3D-SMSN imaging within tissues. This development allowed us to image through 30-μm-thick brain sections to visualize and reconstruct the morphology and the nanoscale details of amyloid-β filaments in a mouse model of Alzheimer's disease