Summary Aging reduces our bodies’ ability to respond to pathogens and vaccines. This is thought to at least in part be due to impaired functions of adaptive immune cells, which are educated and activated in the lymph nodes. The lymph nodes are highly organized tissues that coordinate T and B cell (the primary adaptive immune cell) functions through a complex system of interactions and migration within the tissue. Recent work has shown that T cell migration and motility is impaired in aging, and that this is correlated to the age of the lymph node tissue specifically, not the age of the T cells. Interestingly, researchers have demonstrated that during aging extracellular matrix materials and adipose tissue is accumulated in the lymph nodes. Changes in the composition of lymph node tissue may result in changes in its biophysical properties like elastic and viscous moduli, as well as stiffness. An increase in stiff adipose tissue and more fibrous material is likely to increase the tissue’s elastic modulus and stiffness. Adaptive immune cells have been shown to respond poorly to increases in stiffness and elastic modulus, as indicated by a reduced ability to migrate in proximity to extracellular matrix materials and impaired activation in stiff environments. Thus far, no studies have been able to correlate these structure-function relationships in the lymph node. This is likely due to the difficult nature of extracting biophysical properties like elastic and loss moduli and stiffness from small tissues, while maintaining tissue integrity, and correlating these properties to different regions of a tissue. We have pioneered the use of multiple particle tracking (MPT) and Brillouin microscopy to study lymph node biophysical properties ex vivo via tissue slice cultures. MPT uses nanoparticle Brownian motion within a tissue to extrapolate its elastic and viscous moduli, pore size, and viscosity and we have used this method to assess biophysical properties of T and B cell regions of skin and mesentery lymph nodes. Brillouin microscopy uses interactions between inherent and incident phonons to calculate a shift that can estimate the uniaxial stress-strain ratio at high frequency, resulting in a heat map of moduli that are correlated with the tissue’s elastic modulus. Here, we will correlate the biophysical properties obtained through both of these methods with extracellular matrix and lymph node compartments using immunofluorescence to provide a complete picture of how the lymph node structure impacts local properties in the context of aging. We will also assess how specific extracellular matrix components, such as different collagens, contribute to lymph node biophysical properties. Altogether, our proposed studies will build key knowledge in our understanding of how lymph node structure affect their biophysical properties and will lay the foundation for future investigations of structure-function relationships between lymph node biophysical properties and...