Development and applications of magneto-optical scanning probe microscopy

Rapid progress in all areas of science on the nanometer size scale has promoted increasing interest in techniques of ultrahigh-resolution microscopy. The purpose of this research has been to develop novel scanning probe microscopes and to apply this new instrumentation to biomedical applications of interest. The majority of this dissertation is concerned with the development of a three dimensional (3-D) magnetic and optical force microscope (3DFM) for measurement of 3-D viscoelastic fields in biological systems. An optical detection system measures the 3-D displacement of the probe (a magnetic bead, which has a diameter of 4.4μm or smaller) with a spatial resolution of a few nanometers and a temporal resolution of 40μs. Fast dynamic optical calibration techniques were developed for calibrating each individual tracked particle using a quadrant photodiode placed in the back-focal plane of a 0.7 NA microscope objective. An active feedback control system keeps the bead inside the center of the tracking laser by moving the sample cell.; Thermal position fluctuations (Brownian motion) of the tracked particle provide spatial and viscoelastic information about the local environment. A comprehensive quantitative analysis of the magnetic forces and optical tracking were performed both in theory and through simulation. These results were compared to the experimental results obtained from the actual instrument. A comprehensive noise analysis was performed on the system to delineate the lower limits on the measured displacements from the tracking algorithm. The magnetic forces generated by the 3-D magnetic manipulator were calibrated using samples of known viscosities (sucrose solutions). These applied magnetic forces can be used for the purpose of both force microscopy and manipulation. Some preliminary results are presented on the measured viscosity of mucus from normal human lung epithelium.; The use of non-invasive magnetic and optical fields should facilitate the study of intercellular organelles and give better insight into the structure of the cortical and internal cytoskeleton. In addition, viscoelastic measurements are of great practical value to quantify the effects of drugs, mutations, and diseases on the mechanical structure of biological cells.