Super Resolution Nanoscopy For Bio-Imaging Based On Cw Laser Sted Microscopy

Optical resolution has always been restricted by the Ernst diffraction limit, which states that lens-based optical microscopes are unable to discern details that are closer together than half the wavelength of light. Until about 20 years ago, it was deemed impossible to see particles, such as proteins, that are less than 100 nanometers apart. However, with advancements in fluorescence microscopy, particularly Stimulated Emission Depletion (STED) microscopy, it is now possible to see such structures at the sub organelle level. Although difficult, the end result is an easy to read STED image. The purpose of this experiment was to determine the position at which the best STED image could be captured, allowing for the clearest possible image. In order to find this ideal position, several stimulation trials were run, and with each trial the piezo scanning stage was only moved a few nanometers. The position of the particle in all three coordinate directions (x, y, z) was recorded and the strength of the UV and green laser was recorded as well. In order to best determine the ideal position, several computer programs were used that produced graphs and images that helped determine the most efficient location for the particle. This experiment resulted in images and graphs that allowed the particle to be seen on the nanoscale level with greater clarity than had been obtained previously. The results for this experiment can be used for a wider variety of applications in bio-imaging and could further enable identification of specific proteins in cells