Characterization of artificial and biological lipid vesicles using TIRF and SPR
Licentiate thesis, 2018
Synthetic lipid vesicles serve as important mimics of cells and the natural membranes that they are enclosed by. As such they are frequently used as simplified models of the highly complex cell membrane to aid in-depth physicochemical and biological characterization of this essential biological structure. Lipid vesicles also fulfill fundamental biological functions, for example in intercellular signaling via extracellular vesicles and also as signal-substance containing intercellular secretory vesicles in the synapses of neurons and in secretory cells of the endocrine glands, which release their cargo to the extracellular space in response to external cues. Lipid-based nanoparticles are also of increasing importance as drug carriers, both for targeted release at specific tissues and for improved cellular uptake.
There are today many techniques available to probe a multitude of lipid vesicle properties, including size, structure, content, molecular composition etc. In this thesis work, we have contributed improved means to quantify vesicle size using fluorescence microscopy by using total internal reflection fluorescence (TIRF) microscopy to correlate the measured distribution in fluorescence intensity of individual vesicles to their size, as measured by nanoparticle particle tracking analysis (NTA). A similar approach has been used before by others, but the formalism used that have won prevalence contains a mathematical error, which motivated the introduction of an improved expression for converting total vesicle intensity to vesicle size. We present the difference between the former and the latter formalism, as well as the possible negative impact of the former when used to draw conclusions for larger sized vesicles in a number of studies. One example when this type of analysis is crucial, is in studies were a certain vesicle property is correlated with vesicle size. One such example is studies of membrane protein function, which is often dependent of membrane curvature.
In the second work, we used surface plasmon resonance (SPR) and amperometry as quantitative methods to investigate whether secretory dense core vesicles, isolated from bovine chromaffin cells from the medulla of adrenal glands, are able to maintain their high loading of catecholamine molecules after vesicle isolation and purification and how the vesicle catecholamine content is affected by vesicle exposure to osmotic stress. We found, as also previously reported by intracellular amperometry measurements in live cells, that dense core vesicles release part of the vesicle catecholamine content when exerted to a hyperosmotic shock, and also that this release occurs very rapidly in response to the applied osmotic stress. This work demonstrates the strength of using different complementary label-free measurements to account for the total number of catecholamine molecules in such vesicles and for monitoring molecule release from vesicle compartments in real-time. Further, by using knowledge gained about chromaffin vesicle size from TEM together with changes in refractive index as probed with SPR using suspensions based on ordinary and heavy water, we could estimate the hydration level of the dense protein core of the chromaffin vesicles.
total internal reflection (TIRF) microscopy
surface plasmon resonance (SPR)
nanoparticle tracking analysis (NTA)
large dense core vesicles (LDCVs)