Optical sectioning microscopy with spatial modulated excitation laser

Abstract

Optical microscopy is one of the most widely used tools in biomedical researches. However, in the observation of thick biological samples, conventional optical microscopes suffer from background from out-of-focus structures. In this dissertation, excitation patterns are designed to improve optical sectioning capacity for imaging systems. First we improved the point scanning microscopy with excitation laser modulations. We used a mirror to reflect the incident laser to produce the interference in the focal region. It is found both the sectioning capacity and peak intensity of excitation and depletion beam in STED are improved and the signal to noise ratio is enhanced. We also developed a sectioning point scanning based phase contrast microscopy: Schlieren confocal microscopy. We found this method could obtain similar effect as the Differential interference microscopy, keeps the optical sectioning capacity of confocal, and is fully compatible with confocal. Furthermore, we applied excitation modulation to inhibit the background in light field microscopy. We combined light sheet illumination with microlens array coupled detection. It is demonstrated this method could significantly reduce the background in conventional light field microscopy and faster speed compared to light sheet. In conclusion, we succeed to apply spatial modulated excitation to point scanning and volume imaging systems, and improved their sectioning capacity. Our research could provide promising tools for in-vivo observations in large specimens.