Adaptive beam control and analysis in fluorescence microscopy

Abstract

This thesis details three novel advances in instrumentation that are each related to performance improvement in wide-field visible-spectrum imaging systems. In each case our solution concerns the assessment and improvement of optical imaging quality. The three instruments are as follows: The first is a portable transmission microscope which is able to correct for artificially induced aberrations using adaptive optics (AO). The specimens and the method of introducing aberrations into the optical system can be altered to simulate
the performance of AO-correction in both astronomical and biological imaging. We present the design and construction of the system alongside before-and-after AO-correction images for simulated astronomical and biological images. The second instrument is a miniature endoscope camera sensor we re-purposed for use as a quantitative beam analysis probe using a custom high dynamic range (HDR) imaging and reconstruction procedure. This allowed us to produce quantitative flux maps of the illumination beam intensity profile within several operational fluorescence microscope systems. The third and final project
in this thesis was concerned with an adaptive modification to the light sheet illumination beam used in light sheet microscopy, specifically for a single plane illumination microscope (SPIM), embracing the trade-off between the thickness of the light sheet and its extent across the detection field-of-view. The focal region of the beam was made as small as possible and then matched to the shape of curved features within a biological specimen by using a spatial light modulator (SLM) to alter the light sheet focal length throughout the vertical span of the sheet. We used the HDR beam profiling camera probe mentioned earlier to
assess the focal shape and quality of the beam. The resulting illumination beam may in the future be used in a modified SPIM system to produce fluorescence microscope images with enhanced optical sectioning of specific curved features.