Two-photon microscopy with beam and wavelength engineering

Abstract

With conventional two-photon microscopy (2PM) that has a short depth of field (DOF), it is usually hard to monitor the dynamics of individual neurons and their networks simultaneously, as they may extend over several hundreds of micrometers volumetrically. Fortunately, with the help of the non-diffracting beams (NDBs), such as the Bessel beam and Airy beam, we can monitor the neuron activities in a projected view of the three-dimensional (3D) volume by scanning the sample with an extended DOF. However, there are several limitations when directly applying the NDBs in 2PM. The first one is the image contrast degradation from the side lobes; the second limitation is the worse lateral resolution of the NDB than the conventional 2PM using the tightly focused Gaussian beam; the third compromise is the lack of axial resolution. Therefore, techniques are required to engineer the NDBs to solve above problems. In addition to the beam engineering, the laser source in a 2PM system is also dominating for imaging performance, especially the excitation wavelength, which corresponds to different fluorescence proteins and second harmonic generation (SHG) process. The wavelength ranging from 850 to 950 nm is significant for 2P imaging. The current two methods to realize this region, including using the neodymium-doped fibers (NDFs) or the frequency shift, both have several limitations. For the former, the desired wavelength from the three-level transition of the NDF is relatively low; for the latter, energetic pump source and specialty fibers are usually required to excite the nonlinearities for wavelength shift. Consequently, efficient approaches with better laser qualities are demanded to realize the ~900-nm region. The aim of this thesis is to optimize the 2PM performance by beam and wavelength engineering. My contributions are divided into three parts: 1) We utilized the 1st- and 3rd-order Bessel beams to improve the resolution and contrast of the Bessel-beam-based 2PM, respectively. The resolution enhancement was based on the smaller dimension of the dark spot from the 1st-order Bessel beam, while the contrast enhancement was attributed to the well-matched lateral intensity distributions of the 3rd-order Bessel beam with the side rings of the 0th-order Bessel beam. The image subtraction methods are easy to be operated, and suitable for diverse imaging platforms and sample types. 2) We utilized mirrored Airy beams (MABs) and Bessel droplet to resolve the imaging depth of the NDB-based 2PM. The reconstruction mechanism of the MABs was the mapping from the lateral displacement to the depth location, while the Bessel droplet was based on the Fourier transform to resolve the depth from the spatial frequency domain. Both techniques have faster speeds to acquire the volumetric information than the conventional Gaussian-beam-based 2PM, while maintaining the diffraction-free nature that is beneficial for deeper penetration. 3) We designed and established two all-fiber mode-locked lasers at ~1780 and ~1870 nm based on thulium-doped fibers, and we utilized the frequency-doubled light centered at 890 and 937 nm for SHG and 2P fluorescence imaging. Abundant biological tissues are imaged to demonstrate practical applications for both lasers.