Abstract
Quantitative phase imaging (QPI) has become a vital tool in bioimaging, offering precise measurements of optical phase and, thus, of key cellular metabolism metrics, such as dry mass and density. However, only a few quantitative phase imaging applications have been demonstrated in optically thick specimens, where scattering increases background and reduces contrast. Building upon the concept of structured illumination interferometry, Gradient Retardance Optical Microscopy (GROM) for quantitative phase imaging of both thin and thick samples was introduced in Chapter 3. GROM transforms any standard Differential Interference Contrast (DIC) microscope into a quantitative phase imaging platform by incorporating a liquid crystal retarder into the illumination path, enabling independent phase-shifting of the DIC microscope's sheared beams. GROM greatly simplifies related configurations, reduces costs, and eradicates energy losses in parallel imaging modalities, such as fluorescence. Among fluorescence imaging modalities, light-sheet microscopy (LSM) stood out by delivering high-contrast volumetric resolution with minimal photodamage. As commonly acknowledged, scattering, absorption, and refractive index inhomogeneities are known to cause a lot of challenges in light-sheet microscopy (LSM). By integrating temporal focusing (TF) and light-sheet microscopy (LSM), a 2-photon fluorescence (2P-FL) microscopy modality named TF-LSM was developed in Chapter 4. TF-LSM possesses such capabilities in addressing challenges that one of them is to see in inhomogeneous media. In the most scattering environments such as uncleared root tissues, imaging with enhanced homogeneity of host and infected fungi structures was demonstrated. In as well as synthetic biofilms, imaging uniformity at various depths was exhibited. Meanwhile its other ability is to see in refractive index (RI)-induced aberrations, where zebra fisheye imaging without corrections was demonstrated for the first time. Besides TF-LSM, spatial wavefront shaping, used to generate lattice and Airy light-sheets, has been particularly effective in advancing light-sheet microscopy (LSM) beyond the Rayleigh limit. Yet, light-sheet microscopy (LSM) remains underutilized, largely due to its rigid dual-objective requirement that complicates sample handling and enforces a trade-off between imaging field of view (FoV) and resolution. Here in Chapter 5, a single-objective strategy that exploits space-time (ST) correlations for the first time, was introduced by the name of space-time light-sheet microscopy (ST-LSM). ST-LSM goes beyond separate spatial or temporal modulation to jointly modulate the spatiotemporal spectral structure of a pulse. Uniquely, this enabled light sheets with wavelength-scale thickness over millimeter-scale distances. When compared to state-of-the-art approaches in its category, ST-LSM eliminates the dual-objective constraint, expands the sample-accessible volume by 25×, and increases the field of view (FoV) by 10× without sacrificing resolution. The versatility of ST-LSM was demonstrated by using a single setup to image specimens across 10^4 of magnitude in size, from whole roots and developing embryos, down to mammalian cells with sub-cellular resolution. ST-LSM was positioned as an accessible and high-performance optical microscopy platform, by these results, at a variety of biological scales.