Imaging of vesicular structure by correlative microscopy

Correlative microscopy is an imaging technique in which optical microscopes and electron microscopes are combined. The most commonly used type of correlative microscopy is the combination of light and electron microscopes. The systems are called Correlative Light and Electron Microscopy (CLEM). Fluorescent microscope is generally used as an optic microscope, in this combination. FM that provides view from a large area of the specimen and gives a general information about localization of interested molecule is fluorescently labelled but FM has no ability to provide high-resolution image as electron microscopy therefore cannot give information about ultra- structure of molecules. Correlative microscopy in which combination of FM that provides imaging of fluorescently labelled molecules and EM that has the unique high resolution capacity, together enables the study of the structure and function of biological specimens at the highest resolution. With development of correlative microscopy, it becomes possible to image proteins and RNAs that are located in or surface of vesicules at high resolution. However, the system still needs to be developed methodologically. Although CLEM have unique properties, it has serious challenges. The most challenging thing is preparation of the specimen. Because steps of sample preparation for FM and TEM are not proper each other. The other challenge is region of interest that is fluorescently labelled for FM, which is hard to trace in the TEM. It is time consuming and also sometimes impossible. Therefore studies of developing protocols for correlative microscopy imaging is ongoing. Exosomes are Nano-sized vesicles of 30-150 nm in size which are enclosed with lipid bilayer. They are released from most type of cells like Erythrocytes, lymphocytes, dendritic cells, epithelial cell, cancer cells and they can be isolated from body fluids like Saliva, plasma, urine, amniotic fluid, bronchial lavage fluid, synovial fluid, breast milk. Exosomes which are identified new mechanism for cell-cell communication, have high potential of biomarker and can be loaded of interested molecules like drugs so they can be used as a biomarker for diagnosis of diseases and as a carrier for therapy of diseases. Although all the known facts about exosomes, there are the unknowns of exosomes are quite a lot. For this reason, visualization of exosomes in high resolution is a very important method to understand their morphology and functions. Contrary to indirect methods, it is a definite proof for assets, locations and behaviors of exosomes. Aim: In this study, exosomes are isolated from HeLa cells and visualize by correlative microscopy. Based on our current literature knowledge, exosomes are not visualized by correlative microscopy before. Method: Exosomes are isolated from HeLa cells and they are labelled with fluorescent conjugated anti-CD63 antibody which is binds surface marker of exosome. Exosome sample is put into special holder. So fluorescent labelled exosomes are visualized by confocal microscope and coordinates of visualized area are saved using Zeiss ZEN Blue software programme. After that exosomes are contrasted for EM imaging. After EM imaging are completed, 3 correlation points are determined between images which come from different microscope system. Then fluorescence image and the ultra-detail image are superimposed by using Zeiss Shuttle & Find software programme. So correlation image is created. Results: Exosomes were labelled with fluorescently conjugated antibodies from the surface marker CD63 protein was visualized by Confocal-SEM and Confocal-TEM combination of Correlative microscopy Conclusion: With this study, exosomes that were isolated from cells were visualized for the first time by correlative microscopy.