| As biological investigations push into the nanoscale regime, the ability to resolve sub-diffraction images is increasingly important. Stimulated emission depletion (STED) microscopy breaks the diffraction barrier by decreasing the excitation spot size via depletion of the peripheral excitation regions. This dissertation presents experimental results throughout the developmental process of building a STED microscope. Objective lens considerations and depletion optimization issues are investigated. Thorough stimulated emission depletion dynamics for 100 nm diameter fluorescent microspheres (Invitrogen FluoSpheres 350/440) is presented. Depletion is characterized with respect to exposure time, emission wavelength, time delay, depletion beam intensity, excitation beam intensity, number of fluorescent microspheres, and beam overlap. One-dimensional sub-diffraction imaging capability is also demonstrated. In addition, prospects for higher resolution capabilities are addressed. The STED microscope built in this work utilizes commercial off-the-shelf photonics components and represents a simpler, less expensive scheme to achieving sub-diffraction capabilities. |
