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We describe the advantages of diffuse illumination in laser holography for ophthalmology. The presence of a diffusing element introduces an angular diversity of the optical radiation and reduces its spatial coherence, which spreads out the energy distribution of the illumination beam in the focal plane of the eyepiece. The field of view of digitally computed retinal images can easily be increased as the eyepiece can be moved closer to the cornea to obtain a Maxwellian view of the retina without compromising ocular safety. Compliance with American and European safety standards for ophthalmic devices is more easily obtained by preventing the presence of a laser hot spot observed in front of the cornea in the absence of a scattering element. Diffuse laser illumination does not introduce any adverse effects on digitally computed laser Doppler images., Comment: 9 pages

Glaucoma remains among the leading causes of blindness despite many treatment options available today. Effective treatment requires early diagnosis, which is difficult to achieve with existing imaging technologies that detect the already inflicted irreversible posterior eye damage. One improved approach could be to detect the changes in trabecular meshwork of the anterior eye that precede the intraocular pressure buildup and vision loss. Here we report a device that enables in vivo exploration of trabecular meshwork at cell resolution. The instrument combines two orthogonal views: en face view of time-domain full-field OCT for high-resolution and cross-sectional view of spectral-domain OCT for aligning to the meshwork. The device resolves micron-level corneoscleral and uveal pores, meshwork beams and internal elastic fibers, ciliary body tendons and trabecular cell nuclei. Capable of detecting blockage of trabecular pores, orthogonal view OCT presents a promising solution to the long-standing problem of early glaucoma diagnosis. Future clinical adoption of the device is facilitated by the compact footprint and broad functionality that includes corneal imaging in healthy subjects and patients (with keratoconus, Fuchs endothelial dystrophy) using the same design.

Effective and accurate in-vivo diagnosis of retinal pathologies requires high performance imaging devices, combining a large field of view and the ability to discriminate the ballistic signal from the diffuse background in order to provide a highly contrasted image of the retinal structures. Here, we have implemented the Partial-Field Illumination Ophthalmoscope, a patterned illumination modality, integrated on a high pixel rate adaptive optics full-field microscope. This non-invasive technique enables us to mitigate the low signal-to-noise ratio, intrinsic of full-field ophthalmoscopes, by partially illuminating the retina with complementary patterns to reconstruct a wide field image. This new modality provides an image contrast spanning from the full-field to the confocal contrast, depending on the pattern size. As a result, it offers various trade-offs in terms of contrast and acquisition speed, guiding the users towards the most efficient system for a particular clinical application., Comment: 6 pages, 5 figures

Laser Doppler holography (LDH) is a full-field blood flow imaging technique able to reveal human retinal and choroidal blood flow with high temporal resolution. We here report on using LDH in the anterior segment of the eye without making changes to the instrument. Blood flow in the bulbar conjunctiva and episclera as well as in corneal neovascularization can be effectively imaged. We additionally demonstrate simultaneous holographic imaging of the anterior and posterior segments by simply adapting the numerical propagation distance to the plane of interest. We used this feature to track the movements of the retina and pupil with high temporal resolution. Finally, we show that the light backscattered by the retina can be used for retro-illumination of the anterior segment. Hence digital holography can reveal opacities caused by absorption or diffusion in the cornea and eye lens.

Retinal degenerative diseases lead to the blindness of millions of people around the world. In case of age-related macular degeneration (AMD), the atrophy of retinal pigment epithelium (RPE) precedes neural dystrophy. But as crucial as understanding both healthy and pathological RPE cell physiology is for those diseases, no current technique allows subcellular in vivo or in vitro live observation of this critical cell layer. To fill this gap, we propose dynamic full-field OCT (D-FFOCT) as a candidate for live observation of in vitro RPE phenotype. In this way, we monitored primary porcine and human stem cell-derived RPE cells in stress model conditions by performing scratch assays. In this study, we quantified wound healing parameters on the stressed RPE, and observed different cell phenotypes, displayed by the D-FFOCT signal. In order to decipher the subcellular contributions to these dynamic profiles, we performed immunohistochemistry to identify which organelles generate the signal and found mitochondria to be the main contributor to D-FFOCT contrast. Altogether, D-FFOCT appears to be an innovative method to follow degenerative disease evolution and could be an appreciated method in the future for live patient diagnostics and to direct treatment choice.

Laser Doppler holography was introduced as a full-field imaging technique to measure blood flow in the retina and choroid with an as yet unrivaled temporal resolution. We here investigate separating the different contributions to the power Doppler signal in order to isolate the flow waveforms of vessels in the posterior pole of the human eye. Distinct flow behaviors are found in retinal arteries and veins with seemingly interrelated waveforms. We demonstrate a full field mapping of the local resistivity index, and the possibility to perform unambiguous identification of retinal arteries and veins on the basis of their systolodiastolic variations. Finally we investigate the arterial flow waveforms in the retina and choroid and find synchronous and similar waveforms, although with a lower pulsatility in choroidal vessels. This work demonstrates the potential held by laser Doppler holography to study ocular hemodynamics in healthy and diseased eyes.

The choroid is a highly vascularized tissues supplying the retinal pigment epithelium and photoreceptors. Its implication in retinal diseases is gaining increasing interest. However, investigating the anatomy and flow of the choroid remains challenging. Here we show that laser Doppler holography provides high contrast imaging of choroidal vessels in humans, with a spatial resolution comparable to state of the art indocyanine green angiography and optical coherence tomography. Additionally, laser Doppler holography contributes to sort out choroidal arteries and veins by using a power Doppler spectral analysis. We thus demonstrate the potential of laser Doppler holography to improve our understanding of the anatomy and flow of the choroidal vascular network.

Laser Doppler holography is a planar blood flow imaging technique recently introduced in ophthalmology to image human retinal and choroidal blood flow non-invasively. Here we present a digital method based on the Doppler spectrum asymmetry that reveals the local direction of blood flow with respect to the optical axis in out-of-plane vessels. This directional information is overlaid on standard grayscale blood flow images to depict flow moving towards the camera in red and flow moving away from the camera in blue, as in ultrasound color Doppler imaging. We show that thanks to the strong contribution of backscattering to the Doppler spectrum in out-of-plane vessels, the local axial direction of blood flow can be revealed with a high temporal resolution, which enables us to evidence pathological blood flow reversals. We also demonstrate the use of optical Doppler spectrograms to quantitatively monitor retinal blood flow reversals.

Structured illumination microscopy (SIM) is one of the most versatile super-resolution techniques. Yet, its application to live imaging has been so far mainly limited to fluorescent and stationary specimens. Here, we present advancements in SIM to jointly tackle all the challenges of imaging living samples, i.e., obtaining super-resolution over an undistorted wide-field while dealing with sample motion, scattering, sample-induced optical aberrations and low signal-to-noise ratio. By using adaptive optics to compensate for optical aberrations and a reconstruction algorithm tailored for a moving and thick tissue, we successfully applied SIM to in vivo retinal imaging and demonstrated structured illumination ophthalmoscopy for high contrast super-resolution in vivo imaging of the human retina., Comment: 14 pages, 8 figures, manuscript under consideration for a peer-reviewed journal

High-resolution optical imaging methods, such as confocal microscopy and full-field optical coherence tomography, capture flat optical sections of the sample. If the sample is curved, the optical field sections through several sample layers and the view of each layer is reduced. Here we present curved-field optical coherence tomography, capable of capturing optical sections of arbitrary curvature. We test the device on a challenging task of imaging the human cornea in vivo and achieve 10x larger viewing area comparing to the clinical state-of-the-art. This enables more precise cell and nerve counts, opening a path to improved diagnosis of corneal and general health conditions (e.g. diabetes). The method is non-contact, compact and works in a single-shot, making it readily available for use in optical research and clinical practice.

We demonstrate the feasibility of high-quality digital holography of the human retina in real-time with a fast camera and commodity computer hardware. High throughput rendering of digital Fresnel holograms from optically-acquired inline interferograms is performed in conjunction with temporal demodulation by projection of hologram sequences onto a data-derived basis in order to discriminate local narrowband coherent detection contrasts, mostly due to blood flow and optical absorption, from spurious interferometric contributions. Digital holograms are calculated from a sustained input stream of 16-bit, 1024-by-1024-pixel interferograms recorded at up to 500 frames per second, processed by principal component analysis. This temporal signal demodulation scheme consists in the projection of stacks of 32 consecutive holograms onto a basis calculated by eigendecomposition of the matrix of their time-lagged covariance; it is performed up to 20 times per second with commodity computer hardware.

Laser Doppler holography (LDH) is an interferometric blood flow imaging technique based on full-field measurements of the Doppler spectrum. LDH has so far been demonstrated in the retina with ultrafast cameras, typically at 75 kHz. We show here that a similar method can be implemented with camera frame rates 10 times slower than before. Thanks to energy conservation, low and high frequency local power Doppler signals have opposite variations, and a simple contrast inversion of the low frequency power Doppler reveals fast blood flow beyond the camera detection bandwidth for conventional laser Doppler measurements. Relevant blood flow variations and color composite power Doppler images can be obtained with camera frame rates down to a few kHz.

Laser Doppler holography (LDH) is a full-field interferometric imaging technique recently applied in ophthalmology to measure blood flow, a parameter of high clinical interest. From the temporal fluctuations of digital holograms acquired at ultrafast frame rates, LDH reveals retinal and choroidal blood flow with a few milliseconds of temporal resolution. However LDH experiences difficulties to detect slower blood flow as it requires to work with low Doppler frequency shifts which are corrupted by eye motion. We here demonstrate the use of a spatio-temporal decomposition adapted from Doppler ultrasound that provides a basis appropriate to the discrimination of blood flow from eye motion. A singular value decomposition (SVD) can be used as a simple, robust, and efficient way to separate the Doppler fluctuations of blood flow from those of strong spatial coherence such as eye motion. We show that the SVD outperforms the conventional Fourier based filter to reveal slower blood flow, and dramatically improves the ability of LDH to reveal vessels of smaller size or with a pathologically reduced blood flow.

Tubular structure tracking is a crucial task in the fields of computer vision and medical image analysis. The minimal paths-based approaches have exhibited their strong ability in tracing tubular structures, by which a tubular structure can be naturally modeled as a minimal geodesic path computed with a suitable geodesic metric. However, existing minimal paths-based tracing approaches still suffer from difficulties such as the shortcuts and short branches combination problems, especially when dealing with the images involving complicated tubular tree structures or background. In this paper, we introduce a new minimal paths-based model for minimally interactive tubular structure centerline extraction in conjunction with a perceptual grouping scheme. Basically, we take into account the prescribed tubular trajectories and curvature-penalized geodesic paths to seek suitable shortest paths. The proposed approach can benefit from the local smoothness prior on tubular structures and the global optimality of the used graph-based path searching scheme. Experimental results on both synthetic and real images prove that the proposed model indeed obtains outperformance comparing with the state-of-the-art minimal paths-based tubular structure tracing algorithms.

Optical tomographic cross-sectional images of biological samples were made possible by interferometric imaging techniques such as Optical Coherence Tomography (OCT). Owing to its unprecedented view of the sample, OCT has become a gold standard, namely for human retinal imaging in the clinical environment. In this Letter, we present Optical Incoherence Tomography (OIT): a completely digital method extending the possibility to generate tomographic retinal cross-sections to non-interferometric imaging systems such as en-face AO-ophthalmoscopes. We demonstrate that OIT can be applied to different imaging modalities using back-scattered and multiply-scattered light including systems without inherent optical sectioning. We show that OIT can be further used to guide focus position when the user is "blind" focusing, allowing precise imaging of translucent retinal structures, the vascular plexuses and the retinal pigment epithelium using respectively split detection, motion contrast, and autofluorescence techniques., Comment: 4 Figures, 3 Videos

Structured Illumination Microscopy (SIM) is an imaging technique for achieving both super-resolution (SR) and optical sectioning (OS) in wide-field microscopy. It consists in illuminating the sample with periodic patterns at different orientations and positions. The resulting images are then processed to reconstruct the observed object with SR and/or OS. In this work, we present BOSSA-SIM, a general-purpose SIM reconstruction method, applicable to moving objects such as encountered in in vivo retinal imaging, that enables SR and OS jointly in a fully unsupervised Bayesian framework. By modeling a 2-layer object composed of an in-focus layer and a defocused layer, we show that BOSSA-SIM is able to jointly reconstruct them so as to get a super-resolved and optically sectioned in-focus layer. The achieved performance, assessed quantitatively by simulations for several noise levels, compares favorably with a state-of-the-art method. Finally, we validate our method on open-access experimental microscopy data., Comment: 17 pages, 9 figures, accepted manuscript for Optics Express

We performed non-invasive video imaging of retinal blood flow in a pigmented rat by holographic interferometry of near-infrared laser light backscattered by retinal tissue, beating against an off-axis reference beam sampled at a frame rate of 39 kHz with a high throughput camera. Local Doppler contrasts emerged from the envelopes of short-time Fourier transforms and the phase of autocorrelation functions of holograms rendered by Fresnel transformation. This approach permitted imaging of blood flow in large retinal vessels (30 microns diameter) over 400 by 400 pixels with a spatial resolution of 8 microns and a temporal resolution of 6.5 ms., Comment: 4 pages, 5 figures, 4 links

We report laser Doppler ophthalmoscopic fundus imaging in the rat eye with near-IR heterodyne holography. Sequential sampling of the beat of the reflected radiation against a frequency-shifted optical local oscillator is made onto an array detector. Wide-field maps of fluctuation spectra in the 10 Hz to 25 kHz band exhibit angiographic contrasts in the retinal vascular tree without requirement of an exogenous marker.