Aberration correction in epi-fluorescence microscopy using unknown speckle illumination

Diffraction-limited imaging in epi-fluorescence microscopy remains a challenge when sample aberrations are present or when the region of interest rests deep within an inhomogeneous medium. Adaptive optics is an attractive solution albeit with limited field of view and requiring relatively complicated systems. Alternatively, reconstruction algorithms have been developed over the years to correct for aberrations. Unfortunately, purely postprocessing techniques tend to be ill-posed and provide only incremental improvements in image quality. Here, we report a computational optical approach using unknown speckle illumination and matched reconstruction algorithm to correct for aberrations and reach or surpass diffraction limited resolution. The data acquisition is performed by shifting an unknown speckle pattern with respect to the fluorescent object. The method recovers simultaneously a high-resolution image, the point spread function of the system that contains the aberrations, the speckle illumination pattern, and the shift positions.

Objective assessment of corneal transparency in the clinical setting with standard SD-OCT devices

PURPOSE: To develop an automated algorithm allowing extraction of quantitative corneal transparency parameters from clinical spectral-domain OCT images. To establish a representative dataset of normative transparency values from healthy corneas. METHODS: SD-OCT images of 83 normal corneas (ages 22-50 years) from a standard clinical device (RTVue-XR Avanti, Optovue Inc.) were processed. A pre-processing procedure is applied first, including a derivative approach and a PCA-based correction mask, to eliminate common central artifacts (i.e., apex-centered column saturation artifact and posterior stromal artifact) and enable standardized analysis. The mean intensity stromal-depth profile is then extracted over a 6-mm-wide corneal area and analyzed according to our previously developed method deriving quantitative transparency parameters related to the physics of light propagation in tissues, notably tissular heterogeneity (Birge ratio; $B_r$), followed by the photon mean-free path ($l_s$) in homogeneous tissues (i.e., $B_r \sim 1$). RESULTS: After confirming stromal homogeneity ($B_r < 10$, IDR: 1.9-5.1), we measured a median $l_s$ of 570 $\mu$m (IDR: 270-2400 $\mu$m). Considering corneal thicknesses, this may be translated into a median fraction of transmitted (coherent) light $T_{coh(stroma)}$ of 51$\%$ (IDR: 22-83$\%$). No statistically significant correlation between transparency and age or thickness was found. CONCLUSIONS: Our algorithm provides robust and quantitative measurement of corneal transparency from standard clinical SD-OCT images. It yields lower transparency values than previously reported, which may be attributed to our method being exclusively sensitive to spatially coherent light. Excluding images with central artifacts wider than 300 $\mu$m also raises our median $T_{coh(stroma)}$ to 70$\%$ (IDR: 34-87$\%$).