Clara Pfäffle

In vivo optical imaging of physiological responses to photostimulation in human photoreceptors

Significance Using a full-field optical coherence tomography system, we measured changes in the time that light requires to pass through photoreceptor outer segments, when the retina is stimulated by a light pulse. This effect can be used to monitor the activity of single cones in the living human eye. Objective monitoring of photoreceptor activity using such intrinsic optical signals could have important diagnostic applications in ophthalmology and neurology and might provide insight to facilitate basic research. Noninvasive functional imaging of molecular and cellular processes of vision may have immense impact on research and clinical diagnostics. Although suitable intrinsic optical signals (IOSs) have been observed ex vivo and in immobilized animals in vivo, detecting IOSs of photoreceptor activity in living humans was cumbersome and time consuming. Here, we observed clear spatially and temporally resolved changes in the optical path length of the photoreceptor outer segment as a response to an optical stimulus in the living human eye. To witness these changes, we evaluated phase data obtained with a parallelized and computationally aberration-corrected optical coherence tomography system. The noninvasive detection of optical path length changes shows neuronal photoreceptor activity of single cones in living human retina, and therefore, it may provide diagnostic options in ophthalmology and neurology and could provide insights into visual phototransduction in humans.

Imaging pulse wave propagation in human retinal vessels using full-field swept-source optical coherence tomography

We demonstrate a new noninvasive method to assess biomechanical properties of the retinal vascular system. Phase-sensitive full-field swept-source optical coherence tomography (PhS-FF-SS-OCT) is used to investigate retinal vascular dynamics at unprecedented temporal resolution. The motion of retinal tissue that is induced by expansion of the vessels therein is measured with an accuracy of about 10 nm. The pulse shapes of arterial and venous pulsations, their temporal delays, as well as the frequency-dependent pulse propagation through the capillary bed, are determined. For the first time, imaging speed and motion sensitivity are sufficient for a direct measurement of pulse waves propagating with more than 600 mm/s in retinal vessels of a healthy young subject.

Aberration-free volumetric high-speed imaging of in vivo retina.

Certain topics in research and advancements in medical diagnostics may benefit from improved temporal and spatial resolution during non-invasive optical imaging of living tissue. However, so far no imaging technique can generate entirely diffraction-limited tomographic volumes with a single data acquisition, if the target moves or changes rapidly, such as the human retina. Additionally, the presence of aberrations may represent further difficulties. We show that a simple interferometric setup–based on parallelized optical coherence tomography–acquires volumetric data with 10 billion voxels per second, exceeding previous imaging speeds by an order of magnitude. This allows us to computationally obtain and correct defocus and aberrations resulting in entirely diffraction-limited volumes. As demonstration, we imaged living human retina with clearly visible nerve fiber layer, small capillary networks, and photoreceptor cells. Furthermore, the technique can also obtain phase-sensitive volumes of other scattering structures at unprecedented acquisition speeds.

Reduction of frame rate in full-field swept-source optical coherence tomography by numerical motion correction [Invited]

Full-field swept-source optical coherence tomography (FF-SS-OCT) was recently shown to allow new and exciting applications for imaging the human eye that were previously not possible using current scanning OCT systems. However, especially when using cameras that do not acquire data with hundreds of kHz frame rate, uncorrected phase errors due to axial motion of the eye lead to a drastic loss in image quality of the reconstructed volumes. Here we first give a short overview of recent advances in techniques and applications of parallelized OCT and finally present an iterative and statistical algorithm that estimates and corrects motion-induced phase errors in the FF-SS-OCT data. The presented algorithm is in many aspects adopted from the phase gradient autofocus (PGA) method, which is frequently used in synthetic aperture radar (SAR). Following this approach, the available phase errors can be estimated based on the image information that remains in the data, and no parametrization with few degrees of freedom is required. Consequently, the algorithm is capable of compensating even strong motion artifacts. Efficacy of the algorithm was tested on simulated data with motion containing varying frequency components. We show that even in strongly blurred data, the actual image information remains intact, and the algorithm can identify the phase error and correct it. Furthermore, we use the algorithm to compensate real phase error in FF-SS-OCT imaging of the human retina. Acquisition rates can be reduced by a factor of three (from 60 to 20 kHz frame rate) with an image quality that is even higher compared to uncorrected volumes recorded at the maximum acquisition rate. The presented algorithm for axial motion correction decreases the high requirements on the camera frame rate and thus brings FF-SS-OCT closer to clinical applications.

Systematic in-vivo investigation of intrinsic optical signals in the photoreceptor outer segment (Conference Presentation)

Full-field-swept-source optical coherence tomography is capable of detecting small morphological changes in the living human eye below sub-wavelength range by evaluating the phases. This is used to obtain intrinsic optical signals originating in the photoreceptor outer segment, spatially resolved to single photoreceptors. These were measured ex-vivo in explanted porcine retina as well as in the living human eye. The obtained signals are related to an increase of the optical path length of the outer segments. However, they give no hint wether they are caused by an actual physical expansion of the outer segments or by a changes in the index of refraction. Therefore, systematical measurements were carried out to determine the physical nature and biochemical source of the observed effects.

Imaging of physiological responses to photostimulation in human photoreceptors with full-field swept-source OCT (Conference Presentation)

The non-invasive measurement of cellular physiological responses to photostimulation in living retina may have significant clinical value and give new insight into the vision process. Optical coherence tomography (OCT) has been reported to detect suitable intrinsic optical signals (IOS) in retinal photoreceptor layers upon their stimulation. Commonly, changes in backscattering intensity were observed ex vivo and immobilized animals in vivo. However, in humans measurements were time-consuming and cumbersome. Promising results were achieved when observing phase signals to detect intrinsic optical signals. But to achieve sufficient phase stability to image an entire area of photoreceptors turned out to be challenging. Here, we report full-field swept-source OCT to be sufficiently stable to detect the phase signals after projecting a stimulation image onto the living human retina. We extracted time-courses and signal dependencies from the measured datasets. For long stimuli, we were even able to assign responses to single cones. This functional imaging of photoreceptor activity could potentially be used to detect loss of photoreceptor function prior to visible morphological changes, which is associated with numerous retinal diseases.

Imaging vascular dynamics in human retina using full-field swept-source optical coherence tomography(Conference Presentation)

We demonstrate a new non-invasive method to assess the functional condition of the retinal vascular system. Phase-sensitive full-field swept-source optical coherence tomography (PhS-FF-SS-OCT) is used to investigate retinal vascular dynamics at unprecedented temporal resolution. Motion of retinal tissue, that is induced by expansion of the vessels therein, is measured with an accuracy of about 10 nm. The pulse shape of arterial and venous pulsation, their temporal delay as well as the frequency dependent pulse propagation through the capillary bed are determined. For the first time, imaging speed and motion sensitivity are sufficient for a direct measurement of pulse waves propagating with more than 600 mm/s in retinal vessels of a healthy young subject.