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Correlative light and electron microscopy : methods of studying neurons
Author
Keshmarathy Sacadevan
Supervisor
VanDongen, Antonius M.
Van Dongen, Hendrika M. A.
Kwek, Leong Chuan
Abstract
A better understanding on how chromatin complexes in the nucleus function requires a clearer visualization of their interactions. Due to the diffraction limit of the light, conventional microscopy techniques1 are unable to resolve these complexes. Super resolution microscopy techniques (such as STED2, SIM3 and STORM4) have bypassed the diffraction limit of light to allow visualization of protein interactions in the range of 15-80 nm in the lateral resolution. The resolution is also limited by the sample thickness and the dye size. Electron microscopy (EM) is able to reveal locations of proteins with nanometer resolution. However, most of the time the harsh sample fixation methods required for EM could compromise the fragile structure of the cell and its nucleus. In this dissertation, transmission electron microscope sample preparation has been employed to correlate EM images with light microscopy images obtained by super-resolution techniques including STED and STORM. The cerebellum from embryonic rats was sectioned and stained with fluorescent DNA5 dyes and imaged using STED and STORM microscopy. The results obtained have shown that sample thickness is an important parameter for achieving an optimal resolution. For the cerebellar nuclei the optimal thickness was between 100 nm and 1 micrometer, depending on the imaging methods. This new approach now allows us to study the chromatin complexes of cerebellar nuclei in greater detail.
Date Issued
2017
Call Number
QP363 Kes
Date Submitted
2017