Gesa Franke

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.

Efficient holoscopy image reconstruction

Holoscopy is a tomographic imaging technique that combines digital holography and Fourier-domain optical coherence tomography (OCT) to gain tomograms with diffraction limited resolution and uniform sensitivity over several Rayleigh lengths. The lateral image information is calculated from the spatial interference pattern formed by light scattered from the sample and a reference beam. The depth information is obtained from the spectral dependence of the recorded digital holograms. Numerous digital holograms are acquired at different wavelengths and then reconstructed for a common plane in the sample. Afterwards standard Fourier-domain OCT signal processing achieves depth discrimination. Here we describe and demonstrate an optimized data reconstruction algorithm for holoscopy which is related to the inverse scattering reconstruction of wavelength-scanned full-field optical coherence tomography data. Instead of calculating a regularized pseudoinverse of the forward operator, the recorded optical fields are propagated back into the sample volume. In one processing step the high frequency components of the scattering potential are reconstructed on a non-equidistant grid in three-dimensional spatial frequency space. A Fourier transform yields an OCT equivalent image of the object structure. In contrast to the original holoscopy reconstruction with backpropagation and Fourier transform with respect to the wavenumber, the required processing time does neither depend on the confocal parameter nor on the depth of the volume. For an imaging NA of 0.14, the processing time was decreased by a factor of 15, at higher NA the gain in reconstruction speed may reach two orders of magnitude.

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.

Common approach for compensation of axial motion artifacts in swept-source OCT and dispersion in Fourier-domain OCT

Swept-source optical coherence tomography (SS-OCT) is sensitive to sample motion during the wavelength sweep, which leads to image blurring and image artifacts. In line-field and full-field SS-OCT parallelization is achieved by using a line or area detector, respectively. Thus, approximately 1000 lines or images at different wavenumbers are acquired. The sweep duration is identically with the acquisition time of a complete B-scan or volume, rendering parallel SS-OCT more sensitive to motion artifacts than scanning OCT. The effect of axial motion on the measured spectra is similar to the effect of non-balanced group velocity dispersion (GVD) in the interferometer arms. It causes the apparent optical path lengths in the sample arm to vary with the wavenumber. Here we propose the cross-correlation of sub-bandwidth reconstructions (CCSBR) as a new algorithm that is capable of detecting and correcting the artifacts induced by axial motion in line-field or full-field SS-OCT as well as GVD mismatch in any Fourier-domain OCT (FD-OCT) setup. By cross-correlating images which were reconstructed from a limited spectral range of the interference signal, a phase error is determined which is used to correct the spectral modulation prior to the calculation of the A-scans. Performance of the algorithm is demonstrated on in vivo full-field SS-OCT images of skin and scanning FD-OCT of skin and retina.

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.

Off-axis full-field swept-source optical coherence tomography using holographic refocusing

We demonstrate a full-field swept-source OCT using an off-axis geometry of the reference illumination. By using holographic refocusing techniques, a uniform lateral resolution is achieved over the measurement depth of approximately 80 Rayleigh lengths. Compared to a standard on-axis setup, artifacts and autocorrelation signals are suppressed and the measurement depth is doubled by resolving the complex conjugate ambiguity. Holographic refocusing was done efficiently by Fourier-domain resampling as demonstrated before in inverse scattering and holoscopy. It allowed to reconstruct a complete volume with about 10μm resolution over the complete measurement depth of more than 10mm. Off-axis full-field swept-source OCT enables high measurement depths, spanning many Rayleigh lengths with reduced artifacts.

High resolution holoscopy

Holoscopy is a new imaging approach combining Digital Holography and Full-field Fourier-domain Optical Coherence Tomography. The interference pattern between the light scattered by a sample and a defined reference wave is recorded digitally. By numerical processing of the recorded interference pattern, the back-scattering field of the sample is reconstructed with a diffraction limited lateral resolution over the whole measurement depth since numerical refocusing overcomes the limitation of the focal depth. We present two setup configurations - a low resolution setup based on a Michelson interferometer and a high resolution setup based on a Mach-Zehnder interferometer. Successful measurements were demonstrated with a numerical aperture (NA) of 0.05 and 0.14, respectively and will be presented. Additionally, the effects of filtering spatial frequencies in terms of separating sample signals from artifacts caused by setup reflections is discussed and its improvement on the image quality is shown.

In-vivo retinal imaging with off-axis full-field time-domain optical coherence tomography.

With a simple setup, mainly composed of a low coherence light source and a camera, full-field optical coherence tomography (FF-OCT) allows volumetric tissue imaging. However, fringe washout constrains its use in retinal imaging. Here, we present a novel motion-insensitive approach to FF-OCT, which introduces path-length differences between the reference and the sample light in neighboring pixels using an off-axis reference beam. The temporal carrier frequency in scanned time-domain OCT is replaced by a spatial carrier frequency. Volumetric in-vivo FF-OCT measurements of the human retina were acquired in only 1.3 s, comparable to the acquisition times of current clinically used OCT devices.

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.

Towards microscopic resolution in holoscopy

Holoscopy is a new imaging approach combining digital holography and full-field Fourier-domain optical coherence tomography. The interference pattern between light scattered by a sample and a defined reference wave is recorded and processed numerically. During reconstruction numerical refocusing is applied, overcoming the limitation of the focal depth and thus a uniform, diffraction limited lateral resolution over the whole measurement depth can be obtained. The advantage of numerical refocusing becomes especially significant for imaging at high numerical apertures (NAs). We use a high-resolution setup based on a Mach-Zehnder interferometer with an high-resolution microscope objective (NA = 0.75). For reliable reconstruction of a sample volume the Rayleigh length of the microscope objective and the axial resolution, given by the spectral range of the light source, need to be matched. For a 0.75 NA objective a tunable light source with a sweeping range of ! 300nm is required. Here we present as a first step a tunable Ti:sapphire laser with a tuning range of 187 nm. By characterizing the spectral properties of the Ti:sapphire laser and determining the axial point spread function we demonstrate the feasibility of this light source for high-resolution holoscopy.