Unraveling the functional signals of rods and cones in the human retina: separation and analysis.

In recent years, optoretinography has become an important functional imaging method for the retina, as light-evoked changes in the photoreceptors have been demonstrated for a large number of different OCT systems. Full-field swept-source optical coherence tomography (FF-SS-OCT) is particularly phase-stable, and it is currently the only technique sensitive enough to detect the smaller functional changes in the inner plexiform layer (IPL). However, the resolution of state-of-the art FF-SS-OCT systems is not high enough to distinguish individual photoreceptors. This makes it difficult to separate rods from cones. In this work, we circumvent this problem by separating the functional changes in rods and cones by their different temporal dynamics to the same light stimulus. For this purpose, a mathematical model was developed that represents the measured signals as a superposition of two impulse responses. The developed model describes the measured data under different imaging conditions very well and is able to analyze the sensitivity and temporal dynamics of the two photoreceptor types separately.

Diffuse-illumination holographic optical coherence tomography.

Holographic optical coherence tomography (OCT) is a powerful imaging technique, but its ability to reveal low-reflectivity features is limited. In this study, we performed holographic OCT by incoherently averaging volumes with changing diffuse illumination of numerical aperture (NA) equal to the detection NA. While the reduction of speckle from singly scattered light is only modest, we discovered that speckle from multiply scattered light can be arbitrarily reduced, resulting in substantial improvements in image quality. This technique also offers the advantage of suppressing noises arising from spatial coherence, and can be implemented with a partially spatially incoherent light source for further mitigation of multiple scattering. Finally, we show that although holographic reconstruction capabilities are increasingly lost with decreasing spatial coherence, they can be retained over an axial range sufficient to standard OCT applications.

Observation of natural flexural pulse waves in retinal and carotid arteries for wall elasticity estimation.

The risk of cardiovascular events is linked to arterial elasticity that can be estimated from the pulse wave velocity. This symmetric wave velocity is related to the wall elasticity through the Moens-Korteweg equation. However, ultrasound imaging techniques need improved accuracy, and optical measurements on retinal arteries produce inconsistent results. Here, we report the first observation of an antisymmetric pulse wave: the flexural pulse wave. An optical system performs in vivo wave velocity measurements on retinal arteries and veins. Velocity estimation ranges between 1 and 10 millimeter per second. The theory of guided waves confirms the existence of this wave mode and its low velocity. Natural flexural waves can also be detected at the bigger scale of a carotid artery using ultrafast ultrasound imaging. This second natural pulse wave has great potential of becoming a biomarker of blood vessel aging.

Phase-Sensitive Measurements of Depth-Dependent Signal Transduction in the Inner Plexiform Layer.

Non-invasive spatially resolved functional imaging in the human retina has recently attracted considerable attention. Particularly functional imaging of bipolar and ganglion cells could aid in studying neuronal activity in humans, including an investigation of processes of the central nervous system. Recently, we imaged the activity of the inner neuronal layers by measuring nanometer-size changes of the cells within the inner plexiform layer (IPL) using phase-sensitive optical coherence tomography (OCT). In the IPL, there are connections between the neuronal cells that are dedicated to the processing of different aspects of the visual information, such as edges in the image or temporal changes. Still, so far, it was not possible to assign functional changes to single cells or cell classes in living humans, which is essential for studying the vision process. One characteristic of signal processing in the IPL is that different aspects of the visual impression are only processed in specific sub-layers (strata). Here, we present an investigation of these functional signals for three different sub-layers in the IPL with the aim to separate different properties of the visual signal processing. Whereas the inner depth-layer, closest to the ganglion cells, exhibits an increase in the optical path length, the outer depth-layer, closest to the bipolar cell layer, exhibits a decrease in the optical path length. Additionally, we found that the central depth is sensitive to temporal changes, showing a maximum response at a stimulation frequency of around 12.5 Hz. The results demonstrate that the signals from different cell types can be distinguished by phase-sensitive OCT.

Retinal blood flow imaging with combined full-field swept-source optical coherence tomography and laser Doppler holography.

Full-field swept-source optical coherence tomography (FF-SS-OCT) and laser Doppler holography (LDH) are two holographic imaging techniques presenting unique capabilities for ophthalmology. We report on interlaced FF-SS-OCT and LDH imaging with a single instrument. Effectively, retinal blood flow and pulsation could be quasi-simultaneously monitored. This instrument holds potential for a wide scope of ophthalmic applications.

Retinal blood flow reversal quantitatively monitored in out-of-plane vessels with laser Doppler holography.

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.

Laser Doppler holography of the anterior segment for blood flow imaging, eye tracking, and transparency assessment.

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.

Reverse contrast laser Doppler holography for lower frame rate retinal and choroidal blood flow imaging.

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. Due 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.

Spatio-temporal filtering in laser Doppler holography for retinal blood flow imaging.

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.

Waveform analysis of human retinal and choroidal blood flow with laser Doppler holography.

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 arteries. This work demonstrates the potential held by laser Doppler holography to study ocular hemodynamics in healthy and diseased eyes.

Choroidal vasculature imaging with laser Doppler holography.

The choroid is a highly vascularized tissue 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.

Off-axis digital holography with multiplexed volume Bragg gratings.

We report on an optical imaging design based on common-path off-axis digital holography, using a multiplexed volume Bragg grating. In the reported method, a reference optical wave is made by deflection and spatial filtering through a volume Bragg grating. This design has several advantages, including simplicity, stability, and robustness against misalignment.