טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIssman Liron
SubjectSelf-Assembly of cell Membrane Shed Microparticles:
a Nanostructural Study by Novel Electron
Microscopy Methodologies
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisors Professor Emeritus Yeshayahu Talmon
Professor Benjamin Brenner
Assistant Professor Anat Aharon
Full Thesis textFull thesis text - English Version


Abstract

Cryogenic-temperature scanning electron microscopy (cryo-SEM) is an excellent technique for imaging liquid and semi-liquid materials of high vapor pressure, which are highly viscous, or contain large (> 0.5 ?m) aggregates, in which nanometric details are to be studied. However, so far there have been no adequate tools for controlled cryo-specimen preparation. The specimen preparation stage is critical, because most of those samples are very sensitive to concentration and temperature changes, leading to nanostructural artifacts in the specimens.


            We designed and built a system for easy and reliable cryo-SEM specimen preparation under controlled conditions of fixed temperature and humidity. We describe this new methodology, and demonstrate its applicability, by showing images of three liquid material systems. In all of the examples given, we demonstrate artifact- and contamination-free specimens, which preserved their native nanostructure. Our new system paves the way for a new methodology for the newly re-emerging field of cryo-SEM.


Microparticles (MPs) are membrane vesicles (100-1000 nm in diameter), shed from cells upon activation or apoptosis. MPs are found in blood circulation under normal physiological conditions; their level increases in a variety of diseases, such as cancer and diabetes, and during pregnancy. MPs bearing tissue factor (TF), the main activator of the coagulation cascade, play a role in pathogenesis of pro-thrombotic state in cancer patients.


Despite increasing scientific and clinical interest, there are no standard procedures for isolation, detection and characterization of MPs. MPs size is at the edge of the detection range of conventional methods. As MPs affect and reflect physiological and pathological states, and since their clinical properties (size, concentration, morphology, biochemical composition and cellular origin) are extremely important, we have established a new methodology centered around nano-imaging, which will open new possibilities in this area, and will provide significant new data on MPs nanostructure, their composition, and function.