Date of Award
Open Access Thesis
College of Engineering and Computing
Conventional lens-based (far-field) fluorescence microscopy is a widely used imaging technique with spatial resolution up to 150–350 nm. However, this technology cannot discern very small structural features, because the spatial resolution is limited by diffraction to about half of the wavelength of light (λ/2，λ is the wavelength of light). Hence, most of the developments in microscopy aim at improving resolution. In the past decades, stimulated emission depletion (STED) microscopy has been developed to bypass the diffraction limit for the application in biological imaging with resolution approaching the nanoscale. The basic principle of STED microscopy is to employ a doughnut-shape laser called the depletion laser which inhibits fluorescence emission and improves the resolution of the focal plane by depleting the peripheral fluorescence. Thereby, STED microscopy avoids the diffraction barrier and improves the spatial resolution. STED microscopy has been widely applied to address many problems in biology with both continuous wave and pulsed wave lasers.
Various fluorescent nanoparticles, therefore, are attractive for far-field super-resolution microscopy. During the past decades, fluorescent nanoparticles have been used as a fluorescent label, fluorescent probe or marker for super-resolution imaging in vitro andvivo. In our study, STED microscopy is one of the breakthrough technologies that belongs to far-field optical microscopy and can reach the nanoscale spatial resolution. We demonstrate a far-field optical microscopy based on pulsed-wave lasers with the violet (405 nm) and green lasers (532 nm) for excitation and STED, respectively. Firstly,fluorescent dye - Coumarin 102 is applied to verify the stability and reliability of the STED microscopy. Then, one suitable nanoparticle is selected from three different kinds of nanoparticles (Silica Nanoparticles-NFv465, flouro-Max blue aqueous fluorescent nanoparticles, light yellow nanoparticles) based on their absorption and depletion spectrum and depletion efficiency under different depletion power. Light yellow fluorescent nanoparticles (LYs) are selected for characterizing the spatial resolution of the STED microscopy. Finally, the laser beams of the STED microscopy are utilized to scan along a glass slide, which is coated with the LYs. A two-dimensional image of the LYs pattern is established and compared with the confocal imaging, indicating that a spatial resolution (approximately 76.02 nm) has been obtained in the STED imaging so far. Even though the resolution of STED microscopy with pulsed-laser has the room to be improved, the present work shows that our lab has successfully built up the STED microscopy with the pulsed-laser.
Wang, Y.(2017). Far-Field Optical Microscopy Based on Stimulated Emission Depletion. (Master's thesis). Retrieved from https://scholarcommons.sc.edu/etd/4138