Volumetric stimulated Raman scattering microscopy

Volumetric optical microscopy has the advantages of quantitative and global measurement of three-dimensional (3D) biological specimens with high spatial resolution and minimum invasion. However, current volumetric imaging technologies based on light transmission, scattering or fluorescence cannot reveal specimen’s chemical distribution that brings insights to study the chemical events in organisms and their metabolism, functionality, and development. Stimulated Raman scattering (SRS) microscopy allowing visualization of chemical contents based on their intrinsic molecular vibrations is an emerging imaging technology to provide rapid label-free volumetric chemical imaging. This dissertation describes three methodologies for developing advanced volumetric SRS imaging technologies to address the challenges of imaging in vivo samples, imaging speed, and axial resolution. In the first methodology, SRS volumetric imaging is enabled by axially scanning the laser foci for sectioning different depth layers. In Chapter 2, we utilize a piezo objective positioner to drive the objective. Combining with the tissue clearance technique, we realize volumetric SRS imaging up to 500 µm depth in brain tissues showing the potential for 3D staining-free histology. The limitations of piezo scanning are slow speed and disturbance to in vivo samples while rapidly scanning the objective. To tackle the limitations, in Chapter 3, we develop a remote-focusing volumetric SRS microscope based on a deformable mirror and adaptive optics optimization, allowing focal scanning without physically moving the objective or sample. We demonstrate in vivo monitoring of chemical penetration in human sweat pores. In the second methodology, instead of axially scanning the laser foci, the SRS volumetric imaging is enabled by projection imaging with extended depth-of-focus (DOF) beams such as Bessel beams and low numerical-aperture beams. The extended DOF beams integrate SRS signals along the propagation direction to form projection images; thus, a single lateral scan obtains the volumetric chemical information, significantly increasing the volumetric imaging speed for measuring chemical content over a large volume. In Chapter 4, we describe a stimulated Raman projection microscope for fast quantitation of chemicals in a 3D volume. However, projection imaging intrinsically loses axial resolution. We addressed the limitation by developing SRS projection tomography. Mimicking computed tomography, the axial information is reconstructed by angle-dependent projection images obtained by sequentially rotating the sample in a capillary glass tube within the SRS focus. Nevertheless, sample rotation is complicated and not compatible with in vivo samples. To address the difficulty, in Chapter 5, we develop tilted-angle-illuminated stimulated Raman projection tomography which utilizes tilted-angle beams with a tilted angle respected to the optical axis of the objective to obtain angle-dependent projections. This scheme is free of sample rotation and enables fast projection scanning for pushing the imaging speed. The calibration approach and vector-field back-projection algorithm are developed for the multi-view tomographic reconstruction. In the third methodology, we improve the spatial resolution in miniature volumetric SRS imaging via the innovation of metasurface photonics. In developing an SRS endoscope for volumetric chemical imaging inside the human body, the axial resolution deteriorates due to chromatic and monochromatic aberrations induced by poorly made miniature objective lenses. In Chapter 6, we develop a silicon metasurface tailored for compensating the phase errors between the pump and Stokes wavelengths of a singlet refractive lens. Integrating the metasurface with the refractive lens, the hybrid achromatic metalens is compact and provides nearly diffraction-limit resolution, demonstrating a way for developing high resolution chemical imaging endoscopy.