At the beginning of 1950, many researchers challenged the possibility to overcome the fundamental Abbe limit. An attempt was made by Giuliano Toraldo di Francia, who showed that the width of the point-spread function can be reduced applying a filtering technique (called apodozation) (1). In 1994, a revolutionary event took place in the field of optical microscopy: Hell described a method for circumventing the light diffraction barrier (2). In this way, details that were not visible in diffraction-limited techniques could be imaged using a fluorescence microscopy. Nowadays, these methods termed Far-field fluorescence microscopy or nanoscopy techniques, has become an indispensable tool for scientist to address important biological and biophysical questions at the single molecule level. To highlight the outstanding importance of such techniques, the Royal Swedish Academy of Sciences awarded Eric Betzig, Stefan W. Hell, and William E. Moerner the Nobel Prize in Chemistry 2014 “for the development of super-resolved fluorescence microscopy”. In addition, several important technical improvements, including confocal laser scanning microscopy (CLSM) (3), multiphoton microscopy, 4Pi (4) and I5M (5) have had an important role in the field of optical microscopy. On the other side, in 2015 Boyden and colleagues developed a new method termed Expansion Microscopy (ExM), which allows expanding uniformly biological samples by increasing the relative distances among fluorescent molecules labelling specific cellular components (6). ExM permits to achieve a lateral resolution of about 65 nm, using a conventional - diffracted microscope. However, all super resolution methods demand a particular attention in the sample preparation. Achieving super resolved images require the optimization of every steps involved in the labelling process, from the expression of a fluorescent proteins to the fixation of the biological samples. In the last years, these labelling strategies have obtained a critical role in the field of fluorescence microscopy. In particular, the design and the localization precision of specific affinity probes are crucial features that can restrict the applicability of these techniques. In this work, several labelling approaches and optimization of different staining protocol for super resolution techniques were addressed. My effort was focused on STED nanoscopy and ExM, and how to optimize the labelling protocol, the fluorophores choice for a high labelling density. The optimization of the steps involved in the labelling processes allows me combining ExM with STED nanoscopy (ExSTED), to enhance the final resolution (7). In addition, these techniques were used to decipher molecular assemblies in the cellular nuclei. In particular, my attention was focused on an important layer termed nuclear envelope (NE) (8). This nuclear region encases the genetic material, maintains the regular shape of the nucleus and regulates the gene expression. NE is composed by two lipid bilayer and different class of proteins, which pass through or are strictly linked to the nuclear membranes. Nuclear pore complexes (NPCs) and nuclear lamins, two classes of proteins belonging to the NE, were investigated in this work. In particular, NPCs was used to evaluate the isotropy and calculate the expansion factor (EF) at the nanoscale level in ExM. In this work, we show that Nup153, a filamentous subunit localized in the nuclear pore basket (9), is a good reporter to verify the isotropy of the expansion process and its quantification. In addition, nuclear lamins, in particular lamin A (LA) and its mutation ΔLA50 (10), were used to investigate the physiological and pathological nuclear membrane invagination in normal and aging cells.
|Titolo della tesi:||The nucleus under the microscope. A biophysical approach.|
|Data di discussione:||26-mar-2019|
|Appare nelle tipologie:||Tesi di dottorato|