Sub-diffraction-limited microscopy with fluorescent nanoparticles

Abstract

Due to the properties of light, optical microscopes cannot resolve images of objects that are smaller than a few hundred nanometers wide. This is known as the diffraction limit. There are several techniques that researchers have used to overcome the diffraction limit. Techniques such as STED, NSOM, STORM, and PALM use fluorescence in clever ways to mark and isolate objects of interest so that images of these objects on the nanoscale can be resolved and studied in greater detail. This thesis provides an exploration into using fluorescent nanoparticles to perform sub-diffraction limit imaging. This method is designed to be both low-tech and cost-effective so that it can be easily implemented at Wheaton College. A microscope was constructed consisting of a camera, a sample stage, optical filters, and a laser used to excite the nanoparticles. Exciting the nanoparticles with a laser causes them to fluoresce and the fluorescence can be imaged with the camera. Images of the nanoparticles at various concentrations both in and out of solution were taken and analyzed. The nanoparticles were blurred out in the images because of their small size but individual particles could be located by fitting a Gaussian model to the pixel values. The locations of the nanoparticles were able to be narrowed down in the sample to regions between 11.2 nm and 64.8 nm wide. This constituted a considerable improvement in resolution from a traditional optical microscope, but results were mixed because individual particles were hard to discern in many of the images. There are improvements to be made but, overall, this research opens the door for nanoscale imaging at Wheaton College.