Aperture correlation microscopy

Abstract

Microscopy as applied to the biological sciences has benefited tremendously in the last 50 years from the invention of optically sectioning microscopes. Many techniques for microscopy developed recently offer super-resolved imaging, but often at the cost of light efficiency, signal, acquisition speed or significant amounts of money. This thesis investigates further development of aperture correlation microscopy: a cheap, light-efficient, wide-field, optically sectioning, video-rate imaging technique until now incapable of super-resolution.

An aperture correlation microscope was constructed, using a geometry consisting of 2 separate cameras permitting a larger field size than previous spinning disk aperture correlation systems. This microscope, operating at 16Hz (limited by the cameras) was then tested by imaging different samples and found to match reasonably well to the theory, while producing very clear images of biological samples.

In order to power this aperture correlation microscope, an image registration algorithm must first register the frames from both cameras. We present a high-speed image registration algorithm suitable for fixed deformations, where a calibration pattern can first be imaged, by leveraging the graphics processing unit. In testing, this registration algorithm is capable of framerates of over 200Hz on modest hardware.

A reflection correlation microscope was also constructed and characterised, operating at an illumination wavelength of 405nm, representing an advance into the UV for this technique. Synchronisation of camera acquisition and disk rotation allow for fast acquisition from a single camera. Measurements of its axial resolution match closely to the theory for 4 different illumination pattern pitches. Z-Stacks of transistor and Photovoltaic cell samples were taken in order to demonstrate this system's viability for imaging semiconductor devices.

Finally, a novel type of aperture correlation microscope capable of super-resolution is designed and characterised, leveraging coherent illumination. It is demonstrated how tailoring the spatial frequencies present in the aperture mask can result in enhancement of high frequency information up to twice the diffraction limit. Transfer functions are measured, and found to match reasonably well with theory. Lateral resolution improvement of 1.5 times over conventional microscopy is seen here, paving the way for a new, highly efficient super-resolving microscope.