Atomik Kuvvet Mikroskobu ile Biyomoleküllerin Doğal Ortamlarında Tayini ve Karakterizasyonu

Abstract

One of the most prominent features of AFM is its ability to operate under aqueous solutions compared to other high resolution microscopes. However, imaging in aqueous solutions is a more difficult process than imaging in ambient conditions, because liquid surface causes refraction of laser beam and vibrating the cantilever becomes more difficult due to acoustical resonances. In addition, sample should be immobilized, air bubbles should be prevented and sealing is essential to avoid liquid loss. In order to avoid these type of obstacles, in the scope of this thesis at first mechanical, optical and electronic optimization studies of AKM, manufactured by Nanomagnetics Scientific Instruments Ltd. Şti., were carried out and two prototypes have been developed in this content . During the development of the first prototype mechanical obstacles were eliminated and micron and submicron scale images of the various biological samples could be recorded in the liquid cell. In these studies, the existing liquid cell , kantilever holder design and materials used were changed in view of the literature. As the second phase of this study, true atomic resolution were obtained in liquid with new developments through optical pathways and improvements in electronics assembly. As a result of this study, kantilever could vibrated and also a resonance curve could be recorded properly in the liquid, avoiding unwanted frequency curves towards the center frequency of the curve. Thus with the developed AKM kantilever was thermally vibrated at its center resonance frequency and noise floors were recorded as 19.24 fm / √Hz, 28.43 fm / √Hz was measured in air and liquid respectively, and high-resolution images were recorded at the atomic scale. In the second and final part of the thesis force spectroscopy studies, that makes AFM a most powerful tool for characterization, were carried out . The interactions between membrane proteins/receptors and their cognate ligands initiate most of the cellular response and structural organization. The understanding of these interactions in single molecule level is one of the most important challenges in molecular biology and biophysics studies. Force spectroscopy is extremely accurate characterization technique that is capable of measuring receptor-ligand interactions at pikonewton levels in appropriate physiological conditions. However measuring those interactions and using AFM tip as a sensor, tip should be modified with various crosslinkers with the help of chemical bonds. In the scope of this thesis AFM tip modification was carried out and calibration tests was carried out by using various methods. Validation studies as streptavidin-biotin specific binding studies were used as templates. These studies with lysozyme imprinted polymer binding between lysozyme have been recorded. As the final stage of the thesis, as an example of AFM biomedical applications, determination of osteosarcoma-like cells elasticity was carried depending on the substrate and incubation days. The elastic modulus and hardness of samples can be assessed by the nano-indentation feature of AFM. With this feature, quantitative information about the mechanics of Saos - II cells was obtained and cell mechanics and elasticity changes based on various parameters could be compared in the scope of this study. To conclude, closed liquid cell AKM was improved with using acoustic osilation and optic light scattering with minimizing noise effects in this study. Single cell force spectroscopy and cell elasticity measurements with nanoindentation were completed with using sensitivity of this system. To be capable of detecting interaction between each other biological molecules in physiological environment with designed closed liquid cell high resolution AFM at nano scale, it can be provided that AFM is usable for fundamental sciences and medicine.