Characterization of artificial and biological lipid vesicles using TIRF and SPR
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)
Chalmers, Fysik, Biologisk fysik
Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment
Journal of Applied Physics,; Vol. 118(2015)
Artikel i vetenskaplig tidskrift
Fathali, H. M., Olsson, T., Zhdanov, V., Hook, F., Cans, A-S. Content and Dense-Core Quantification of Chromaffin Vesicles using Surface Plasmon Resonance
Nanovetenskap och nanoteknik (2010-2017)
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Chalmers tekniska högskola
Opponent: Björn Åkerman, Biträdande professor Fysikalisk Kemi, Kemi och Kemiteknik, Chalmers Tekniska Högskola, Sverige