No abstract

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.

Abstract. Nearly all dietary lipids are transported from the intestine to venous circulation through the lymphatic system, yet the mechanisms that regulate this process remain unclear. Elucidating the mechanisms involved in the functional response of lymphatics to changes in lipid load would provide valuable insight into recent implications of lymphatic dysfunction in lipid related diseases. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. The imaging platform provides the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. Utilizing post-acquisition image processing algorithms, we can quantify correlations between vessel pump function, lymph flow, and lipid concentration of mesenteric lymphatic vessels in situ. All image analysis is automated with customized LabVIEW virtual instruments; local flow is measured through lymphocyte velocity tracking, vessel contraction through measurements of the vessel wall displacement, and lipid uptake through fluorescence intensity tracking of an orally administered fluorescently labelled fatty acid analogue, BODIPY FL C 16 . This system will prove to be an invaluable tool for scientists studying intestinal lymphatic function in health and disease, and those investigating strategies for targeting the lymphatics with orally delivered drugs to avoid first pass metabolism.

The recent advances in our understanding of lymphatic physiology and the role of the lymphatics in actively regulating fluid balance, lipid transport, and immune cell trafficking has been furthered in part through innovations in imaging, tissue engineering, quantitative biology, biomechanics, and computational modeling. Interdisciplinary and bioengineering approaches will continue to be crucial to the progression of the field, given that lymphatic biology and function are intimately woven with the local microenvironment and mechanical loads experienced by the vessel. This is particularly the case in lymphatic diseases such as lymphedema where the microenvironment can be drastically altered by tissue fibrosis and adipocyte accumulation. In this review we will highlight contributions engineering and mechanics have made to lymphatic physiology and will discuss areas that will be important for future research.

The pumping innate to collecting lymphatic vessels routinely exposes the endothelium to oscillatory wall shear stress and other dynamic forces. However, studying the mechanical sensitivity of the lymphatic endothelium remains a difficult task due to limitations of commercial or custom systems to apply a variety of time-varying stresses in vitro. Current biomechanical in vitro testing devices are very expensive, limited in capability, or highly complex; rendering them largely inaccessible to the endothelial cell biology community. To address these short-comings, the authors propose a reliable, low-cost platform for augmenting the capabilities of commercially available pumps to produce a wide variety of flow rate waveforms. In particular, the Arduino Uno, a microcontroller development board, is used to provide open-loop control of a digital peristaltic pump using precisely-timed serial commands. In addition, the flexibility of this platform is further demonstrated through its support of a custom-built cell-straining device capable of producing oscillatory strains with varying amplitudes and frequencies. Hence, this microcontroller development board is shown to be an inexpensive, precise, and easy-to-use tool for supplementing in vitro assays to quantify the effects of biomechanical forces on lymphatic endothelial cells.