Phase sensitivity in differential phase contrast microscopy: limits and strategies to improve it

The phase sensitivity limit of Differential Phase Contrast (DPC) with partially coherent light is analyzed in details. The parameters to tune phase sensitivity, such as the diameter of illumination, the numerical aperture of the objective, and the noise of the camera are taken into account to determine the minimum phase contrast that can be detected. We found that a priori information about the sample can be used to fine-tune these parameters to increase phase contrast. Based on this information, we propose a simple algorithm to predict phase sensitivity of a DPC setup, which can be performed before the setup is built. Experiments confirm the theoretical findings. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreemen

Overcoming the resolution limit in retinal imaging using the scattering properties of the sclera

In-vivo imaging of the eye's fundus is widely used to study eye's health. State of the art Adaptive Optics devices can resolve features up to a lateral resolution of 1.5 um. This resolution is still above what is needed to observe subcellular structures such as cone cells (1-1.25 um diameter). This limit in resolution is due to the small numerical aperture of the eye when the pupil is fully dilated (max 0.24). In our work, we overcome this limit using a non-standard illumination scheme. A laser beam is shined on the lateral choroid layer, whose scattered light is illuminating the eye's fundus. Thanks to a Spatial Light Modulator the scattered light from the choroid layer can be manipulated to produce a scanning focus spot on the fundus. The intensity of the reflected light from the fundus is collected from the pupil and used for reconstructing the image

Fully automated detection, segmentation, and analysis of in vivo RPE single cells

Objective To develop a fully automated method of retinal pigmented epithelium (RPE) cells detection, segmentation and analysis based on in vivo cellular resolution images obtained with the transscleral optical phase imaging method (TOPI). Methods Fourteen TOPI-RPE images from 11 healthy individuals were analysed. The developed image processing method encompassed image filtering and normalisation, detection and removal of blood vessels, cell detection and cell membrane segmentation. The produced measures were cellular density of RPE layer, cell area, number of neighbouring cells, eccentricity, circularity and solidity. In addition, we proposed coefficient of variation (CV) of RPE cellular membrane (CMDCV) and the solidity of the RPE cell membrane-shape as new metrics for the assessment of RPE single cells. Results The observed median cellular density of the RPE layer was 3743 cells/mu m(2)(interquartile rate (IQR) 1687), with a median observed RPE cell area of 193 mu m(2)(IQR 141). The mean number of neighbouring cells was 5.22 (standard deviation (SD) 0.05) per RPE cell. The mean RPE cell eccentricity was 0.67 (SD 0.02), median circularity 0.83 (IQR 0.01), and median solidity 0.92 (IQR 0.00). The median CMD(CV)was 0.19 (IQR 0.02). The method is characterised by a median image processing and analysis time of 48 sec (IQR 12) per image. Conclusions The present study provides the first fully automated quantitative assessment of human RPE single cells in vivo. The method provides a baseline for future research in the field of clinical ophthalmology, enabling characterisation and diagnostics of retinal diseases at the single-cell level

Optical phase contrast imaging of human retinal cells by changing the tissue refractive index

Purpose : Based on oblique partially coherent illumination of transparent samples, we developed a simple custom Optical Phase Imaging (OPI) microscope providing a label-free, semi-quantitative phase contrast imaging. The aim of this study was to explore this ex-vivo modality for retinal imaging and correlate it with standard clinical images and fluorescence microscopy. Methods : Multimodal macular imaging was performed on the flat-mounted retina of an eye presenting an epiretinal membrane with cystoid macular edema, enucleated for a peripheral melanoma. After glial fibrillary acidic protein (GFAP) - aquaporin (AQP)-4 – collagen (Col)-IV co-immuno-labeling and nuclei staining, the retina was cleared by index matching in a medium of refractive index (RI) 1.46 to decrease scattering for high-resolution deep-tissue ex vivo imaging. We performed a comparison of the clinical examinations obtained by Optical Coherence Tomography-Angiography and fluorescein angiography before enucleation, with the images obtained with confocal microscopy and OPI microscopy. Ex-vivo imaging of the retina mounted in a medium with a lower RI (1.40), close to the mean RI of Muller glial cell (MGC), was then repeated to better view the latter cells. Results : The retinal vessels were used as landmarks for correlating all imaging modalities. OPI microscopy allowed for different contrast imaging depending on the RI of the mounting medium. With the high RI medium (1.46), deep contrast imaging of nuclei and intraretinal cysts was obtained. The solution with a RI of 1.4 provided an improvement in the contrast of the retinal structures, from the inner layer (AQP4-positive MGC, epi-retinal membrane, nerve fibers surrounded by GFAP-positive astrocytes) to the photoreceptor segments. No AQP4 labeling was observed inside the cyst. AQP4-positive, GFAP-negative cells were visualized on the ColIV-labeled epi-retinal membrane, demonstrating that the membrane is made of retinal Muller glial cells. Conclusions : This morphological correlative imaging study demonstrated OPI on numerous cellular structures of a human retina by tuning the tissue RI. This label-free in-depth imaging modality offers a new research tool to study the cellular origin of retinal diseases

Human retinal pigment epithelium cells can be imaged in vivo with a novel adaptive optics camera using transscleral illumination

Histopathology studies described morphological changes of the retinal pigment epithelium (RPE) specific to the onset and progression of retinal diseases in human eyes. However to date, no valuable imaging tool is used in the clinic to image RPE cells. Transscleral Optical Imaging (TOI) uses an oblique illumination of the fundus combined with adaptive optics to provide cell-resolution images of the retinal layers up to the RPE, in vivo. A prospective study was carried out to assess safety and repeatability of TOI technology and characterize healthy RPE cells