Hendrik Spahr

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.

Full-field speckle interferometry for non-contact photoacoustic tomography

A full-field speckle interferometry method for non-contact and prospectively high speed Photoacoustic Tomography is introduced and evaluated as proof of concept. Thermoelastic pressure induced changes of the objects topography are acquired in a repetitive mode without any physical contact to the object. In order to obtain high acquisition speed, the object surface is illuminated by laser pulses and imaged onto a high speed camera chip. In a repetitive triple pulse mode, surface displacements can be acquired with nanometre sensitivity and an adjustable sampling rate of e.g. 20 MHz with a total acquisition time far below one second using kHz repetition rate lasers. Due to recurring interferometric referencing, the method is insensitive to thermal drift of the object due to previous pulses or other motion. The size of the investigated area and the spatial and temporal resolution of the detection are scalable. In this study, the approach is validated by measuring a silicone phantom and a porcine skin phantom with embedded silicone absorbers. The reconstruction of the absorbers is presented in 2D and 3D. The sensitivity of the measurement with respect to the photoacoustic detection is discussed. Potentially, Photoacoustic Imaging can be brought a step closer towards non-anaesthetized in vivo imaging and new medical applications not allowing acoustic contact, such as neurosurgical monitoring or burnt skin investigation.

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.

Imaging of photothermal tissue expansion via phase sensitive optical coherence tomography

Phase sensitive OCT enables the measurement of thermal expansion in laser irradiated material at high lateral and temporal resolution. In principle, a calculation of the 3D temperature distribution and its temporal evolution should be possible by evaluating the local expansion. This could be utilized for a non-invasive and very fast temperature measurement, e.g. to realize an online dosimetry for photocoagulation. The possibilities of quantitative investigations at high axial and lateral resolution are demonstrated by imaging the reversible thermal expansion in laser irradiated multilayer silicone phantoms.

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.

Coherence and diffraction limited resolution in microscopic OCT by a unified approach for the correction of dispersion and aberrations

Optical coherence tomography (OCT) images scattering tissues with 5 to 15 μm resolution. This is usually not sufficient for a distinction of cellular and subcellular structures. Increasing axial and lateral resolution and compensation of artifacts caused by dispersion and aberrations is required to achieve cellular and subcellular resolution. This includes defocus which limit the usable depth of field at high lateral resolution. OCT gives access the phase of the scattered light and hence correction of dispersion and aberrations is possible by numerical algorithms. Here we present a unified dispersion/aberration correction which is based on a polynomial parameterization of the phase error and an optimization of the image quality using Shannon’s entropy. For validation, a supercontinuum light sources and a costume-made spectrometer with 400 nm bandwidth were combined with a high NA microscope objective in a setup for tissue and small animal imaging. Using this setup and computation corrections, volumetric imaging at 1.5 μm resolution is possible. Cellular and near cellular resolution is demonstrated in porcine cornea and the drosophila larva, when computational correction of dispersion and aberrations is used. Due to the excellent correction of the used microscope objective, defocus was the main contribution to the aberrations. In addition, higher aberrations caused by the sample itself were successfully corrected. Dispersion and aberrations are closely related artifacts in microscopic OCT imaging. Hence they can be corrected in the same way by optimization of the image quality. This way microscopic resolution is easily achieved in OCT imaging of static biological tissues.

In vivo full-field time-domain optical coherence tomography using a spatially coherent off-axis reference (Conference Presentation)

Time domain OCT measures the interference between sample and reference radiation as a function of the reference arm length. In full-field-OCT (FF-OCT) a camera is used instead of a scanned beam for a parallel detection of the interference pattern and thus acquiring a complete en face image. Because multiple images have to be acquired to resolve the phase ambiguity, this method is prone to motion artifacts. We present a novel motion-insensitive approach to FF-OCT. Spatially coherent illumination and an off-axis reference beam is used to introduce path-length differences between reference and sample light in neighboring pixels. This spatial carrier frequency replaces the temporal carrier frequency in scanned TD-OCT. The setup is based on a Mach-Zehnder interferometer with a super-luminescent diode and a CMOS area camera. The Sensitivity of the system was determined to be 75 dB. The field of view was 1.42 x 1.42 mm. Each frame had 237x237 lateral channels at an axial resolution of 9 µm in tissue. By step-wise changing the length of the reference arm between the en face scans, volumetric in vivo FF-OCT measurements of the human retina have been acquired within 1.3 s. OCT with a spatially coherent off-axis reference beam is suitable for in vivo imaging of human retina. The quality of the images is sufficient to discriminate the different tissue layers.