טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBiran Idan
SubjectImmuno-Gold Labeling in the Liquid Phase for
Cryogenic-Temperature Electron Microscopy
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Professor Emeritus Yeshayahu Talmon
Full Thesis textFull thesis text - English Version


Abstract

Liposomes are spheroidal vesicles, composed of one or more lipid bilayers. They serve as a realistic model of bio-membranes; they can be formed in few simple steps, showing the physical principles self-organization processes observed in membranes. Lipid segregation into membrane domains takes part in many cellular processes, such as signal transduction pathways, intracellular trafficking processes, and viral entry and exit from cells. Learning and identifying those domains is of great importance.


Extracellular vesicles (EVs) are nanometric vesicles, ranging in size from several tens of nanometers to more than 1 μm in size. They may originate from the cell membrane (“microparticles”), or from the endosomal system (“exosomes”). Although they are found in healthy physiological conditions, their concentration increases significantly during certain pathologies, such as Crohn's disease, diabetes, and cancer. Most currently available techniques used for the characterization of EVs provide information on EV populations, but they lack in morphological characterization on the single-vesicle level, and also do not give a clear differentiation between EVs types.


We used cryogenic-temperature electron microscopy (cryo-EM), with liquid-phase immuno-gold labelling, to study liposomes, liposomes with α-hemolysin protein and EVs in the liquid phase without chemical fixation. Cryogenic-temperature scanning electron microscopy (Cryo-SEM) was used to image leukemic monocytes (THP1) for high-resolution information of the cell membrane and cell organelles. We used cryogenic-temperature transmission electron microscopy (cryo-TEM) to image the labeled liposomes or EVs, shed upon THP-1 cell stimulation by starvation, or incubation with lipopolysaccharide (LPS).

One of the major challenges of electron microscopy of soft matter in general, and biological systems in particular, is the lack of inherent contrast. That can be overcome by selective attachment of gold nanoparticles, which have high electron density, to specific sites of the object studied. Quite often the selectivity is based on immunological attachment. This so-called immuno-gold labeling is usually performed in the dry state. Our goal has been to develop liquid-phase immuno-gold labelling, thus preserving much better the system native nanostructure.

Cryo-SEM of THP1 cells also revealed distinctive EVs shedding under LPS stimulation. EVs were isolated from the shedding cells, and characterized by cryo-TEM complemented by nanoparticle tracking analysis (NTA) to give quantitative and morphological information. Although morphology differences by cryo-TEM were not visible to us, NTA indicated differences in size and concentration following the different stimulations. LPS was found to have an intensifying effect on cellular shedding, while starvation had reduced effect on shedding in comparison to unstimulated cells. Cryo-TEM and immuno-gold labeling of different molar ratios of mixed phosphatidylcholine (PC) and phosphatidylserine (PS) liposomes was also performed in an attempt to identify lipids rafts.  We showed that Annexin-V specificity in the presence of Ca to PS was an appropriate tool for that goal. Although distinct lipid domains were not visible, we concluded that as we increased the molar ratio of PS in the model liposomes, gold labeling increased as well.

We also attempted to immuno-gold label the cells, and later, after EVs isolation, tried to differentiate between microparticles and exosomes.