Open Collections

Wu, Wentao

University of British Columbia

Multiphoton microscopy (MPM) is an optical microscopy technique visualizing tissues non-invasively in high resolution. MPM utilizes femtosecond laser pulses for multiphoton excitation. MPM provides tissue-specific contrasts that are useful in clinical applications. Two-photon excitation fluorescence (2PEF) visualizes cells; second harmonic generation (SHG) visualizes collagen fibers; third harmonic generation (THG) visualizes lipids. While being utilized in research for decades, clinical applications are still limited. The bulkiness and complexity of free-space lasers, microscope platforms, and scanners prevent convenient clinical use. Different from ex-vivo imaging where labeling can enhance signals, clinical imaging relies on label-free signals which can be significantly weaker. Multimodal MPM with multiple contrasts from two- and three-photon excitations helps clinical imaging enhance biochemical specificity and provide complementary information. Depth-scanning further provides volumetric information about tissue. A handheld MPM imaging head with multimodal and depth-scanning capabilities is needed to carry the technique to clinical applications. This dissertation explores the potential of developing compact depth-scanning multimodal MPM with simultaneous two- and three-photon imaging. Harmonic generation is theoretically analyzed under focused Gaussian beam. The impact of phase-matching is discussed to show interface-sensitivity of SHG and THG. A 1580-nm Er-doped femtosecond fiber laser serves as a compact source for excitation, and part of the laser is converted to 790 nm to achieve dual-wavelength two- and three-photon excitation. Signals of 2PEF, SHG, and THG are separated and then detected by photomultiplier tubes. A signal subtraction method is proposed to address crosstalk. To further improve the compactness, a miniaturized depth-scanning objective (numerical aperture ~0.53) consisting of an aspheric and a plano-convex lens is fabricated. A shape-memory-alloy actuator actuates the aspheric lens for depth scanning over a range of 370 μm. A denoising algorithm is developed to improve the quality of weak-signal images. The denoising method is demonstrated on weak-signal images and compared with other denoising methods, showing outperforming results. Information-rich images containing 2PEF, SHG, and THG contrasts are acquired label-free from animal, human, and plant tissues at various depths up to >200 μm. The performance is evaluated with advantages and limitations discussed. Through this study, the compact depth-scanning multimodal MPM shows great potential for clinical applications.

Text

2024-08-21

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88990

Electrical and Computer Engineering

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/