Ultra-Widefield OCT Angiography.

Optical Coherence Tomography Angiography (OCTA), a functional extension of OCT, has the potential to replace most invasive fluorescein angiography (FA) exams in ophthalmology. So far, OCTA's field of view is however still lacking behind fluorescence fundus photography techniques. This is problematic, because many retinal diseases manifest at an early stage by changes of the peripheral retinal capillary network. It is therefore desirable to expand OCTA's field of view to match that of ultra-widefield fundus cameras. We present a custom developed clinical high-speed swept-source OCT (SS-OCT) system operating at an acquisition rate 8-16 times faster than today's state-of-the-art commercially available OCTA devices. Its speed allows us to capture ultra-wide fields of view of up to 90 degrees with an unprecedented sampling density and hence extraordinary resolution by merging two single shot scans with 60 degrees in diameter. To further enhance the visual appearance of the angiograms, we developed for the first time a three-dimensional deep learning based algorithm for denoising volumetric OCTA data sets. We showcase its imaging performance and clinical usability by presenting images of patients suffering from diabetic retinopathy.

Optical coherence tomography.

Optical coherence tomography (OCT) is a non-contact method for imaging the topological and internal microstructure of samples in three dimensions. OCT can be configured as a conventional microscope, as an ophthalmic scanner, or using endoscopes and small diameter catheters for accessing internal biological organs. In this Primer, we describe the principles underpinning the different instrument configurations that are tailored to distinct imaging applications and explain the origin of signal, based on light scattering and propagation. Although OCT has been used for imaging inanimate objects, we focus our discussion on biological and medical imaging. We examine the signal processing methods and algorithms that make OCT exquisitely sensitive to reflections as weak as just a few photons and that reveal functional information in addition to structure. Image processing, display and interpretation, which are all critical for effective biomedical imaging, are discussed in the context of specific applications. Finally, we consider image artifacts and limitations that commonly arise and reflect on future advances and opportunities.

Dynamic real-time subtraction of stray-light and background for multiphoton imaging.

We introduce a new approach to reduce uncorrelated background signals from fluorescence imaging data, using real-time subtraction of background light. This approach takes advantage of the short fluorescence lifetime of most popular fluorescent activity reporters, and the low duty-cycle of ultrafast lasers. By synchronizing excitation and recording, laser-induced multiphoton fluorescence can be discriminated from background light levels with each laser pulse. We demonstrate the ability of our method to - in real-time - remove image artifacts that in a conventional imaging setup lead to clipping of the signal. In other words, our method enables imaging under conditions that in a conventional setup would yield corrupted data from which no accurate information can be extracted. This is advantageous in experimental setups requiring additional light sources for applications such as optogenetic stimulation.

En face optical coherence tomography: a technology review [Invited].

A review on the technological development of en face optical coherence tomography (OCT) and optical coherence microscopy (OCM) is provided. The terminology originally referred to time domain OCT, where the preferential scanning was performed in the en face plane. Potentially the fastest realization of en face image recording is full-field OCT, where the full en face plane is illuminated and recorded simultaneously. The term has nowadays been adopted for high-speed Fourier domain approaches, where the en face image is reconstructed from full 3D volumes either by direct slicing or through axial projection in post processing. The success of modern en face OCT lies in its immediate and easy image interpretation, which is in particular of advantage for OCM or OCT angiography. Applications of en face OCT with a focus on ophthalmology are presented. The review concludes by outlining exciting technological prospects of en face OCT based both on time as well as on Fourier domain OCT.

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy.

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.

Comparative study between a spectral domain and a high-speed single-beam swept source OCTA system for identifying choroidal neovascularization in AMD.

This comparative study between a SD- and SS-OCTA system for visualizing neovascular patterns in AMD, also assessed the influence of cataract on OCTA imaging. 25 eyes with active CNV (AMD) were documented by FA, ICGA and SD-OCT. Two OCTA devices were used: A custom built SS-OCTA (1050 nm, 400,000 A-scans/s, 5 × 5 mm, no image segmentation); AngioVue (OptoVue, CA, USA) SD-OCTA (840 nm, 70.000 A-scans/s, 3 × 3 mm, SSADA technology). Two retina experts graded CNV types and vascular patterns. Cataract influence on OCTA image quality was reported for the superficial retinal plexus (6 eyes). The SS-OCTA prototype showed more CNV lesions compared to the SD-OCTA system (p = 0.01). Overall sensitivity of SD- and SS-OCTA systems to detect CNV lesions was.32 and.68, respectively. The SS-OCTA system was able to detect discrete lesion characteristics better than the SD-OCTA. No significant difference was found in the ability to identify CNV in treatment-naïve eyes. There was no significant influence of cataract. The SS-OCTA prototype detected CNV-associated vascular patterns more reliably than the SD-OCTA system. This is attributed to the SS-OCTA system's longer center wavelength and higher A-scan rate yielding higher definition and contrast of small neovascular structures. The SS-OCTA system used showed no advantage regarding cataract influence.

Akinetic all-semiconductor programmable swept-source at 1550 nm and 1310 nm with centimeters coherence length.

We demonstrate, for the first time, OCT imaging capabilities of a novel, akinetic (without any form of movement in the tuning mechanism), all-semiconductor, all-electronic tunable, compact and flexible swept source laser technology at 1550 nm and 1310 nm. To investigate its OCT performance, 2D and 3D ex vivo and in vivo OCT imaging was performed at different sweep rates, from 20 kHz up to 200 kHz, with different axial resolutions, about 10 µm to 20 µm, and at different coherence gate displacements, from zero delay to >17 cm. Laser source phase linearity and phase repeatability standard deviation of <2 mrad (<160 pm) were observed without external phase referencing, indicating that the laser operated close to the shot noise limit (~2 × factor); constant percentile wavelengths variations of sliding RIN and ortho RIN <0.2% could be demonstrated, ~5 times better as compared to other swept laser technologies.

In vivo imaging of murine endocrine islets of Langerhans with extended-focus optical coherence microscopy.

AIMS/HYPOTHESIS: Structural and functional imaging of the islets of Langerhans and the insulin-secreting beta cells represents a significant challenge and a long-lasting objective in diabetes research. In vivo microscopy offers a valuable insight into beta cell function but has severe limitations regarding sample labelling, imaging speed and depth, and was primarily performed on isolated islets lacking native innervations and vascularisation. This article introduces extended-focus optical coherence microscopy (xfOCM) to image murine pancreatic islets in their natural environment in situ, i.e. in vivo and in a label-free condition. METHODS: Ex vivo measurements on excised pancreases were performed and validated by standard immunohistochemistry to investigate the structures that can be observed with xfOCM. The influence of streptozotocin on the signature of the islets was investigated in a second step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo. RESULTS: xfOCM circumvents the fundamental physical limit that trades lateral resolution for depth of field, and achieves fast volumetric imaging with high resolution in all three dimensions. It allows label-free visualisation of pancreatic lobules, ducts, blood vessels and individual islets of Langerhans ex vivo and in vivo, and detects streptozotocin-induced islet destruction. CONCLUSIONS/INTERPRETATION: Our results demonstrate the potential value of xfOCM in high-resolution in vivo studies to assess islet structure and function in animal models of diabetes, aiming towards its use in longitudinal studies of diabetes progression and islet transplants.

Laser Doppler imaging for intraoperative human brain mapping.

The identification and accurate location of centers of brain activity are vital both in neuro-surgery and brain research. This study aimed to provide a non-invasive, non-contact, accurate, rapid and user-friendly means of producing functional images intraoperatively. To this end a full field Laser Doppler imager was developed and integrated within the surgical microscope and perfusion images of the cortical surface were acquired during awake surgery whilst the patient performed a predetermined task. The regions of brain activity showed a clear signal (10-20% with respect to the baseline) related to the stimulation protocol which lead to intraoperative functional brain maps of strong statistical significance and which correlate well with the preoperative fMRI and intraoperative cortical electro-stimulation. These initial results achieved with a prototype device and wavelet based regressor analysis (the hemodynamic response function being derived from MRI applications) demonstrate the feasibility of LDI as an appropriate technique for intraoperative functional brain imaging.

Image formation in fluorescence coherence-gated imaging through scattering media.

Recently, we have experimentally demonstrated a new form of cross-sectional, coherence-gated fluorescence imaging referred to as SD-FCT ('spectral-domain fluorescence coherence tomography'). Imaging in SD-FCT is accomplished by spectrally detecting self-interference of the spontaneous emission of fluorophores, thereby providing depth-resolved information on the axial positions of fluorescent probes. Here, we present a theoretical investigation of the factors affecting the detected SD-FCT signal through scattering media. An imaging equation for SD-FCT is derived that includes the effects of defocusing, numerical-aperture, and the optical properties of the medium. A comparison between the optical sectioning capabilities of SD-FCT and confocal microscopy is also presented. Our results suggest that coherence gating in fluorescence imaging may provide an improved approach for depth-resolved imaging of fluorescently labeled samples; high axial resolution (a few microns) can be achieved with low numerical apertures (NA<0.09) while maintaining a large depth of field (a few hundreds of microns) in a relatively low scattering medium (6 mean free paths), whereas moderate NA's can be used to enhance depth selectivity in more highly scattering biological samples.

Extended focus depth for Fourier domain optical coherence microscopy.

We report on a new detection scheme for Fourier domain optical coherence microscopy that exhibits high transverse resolution along an axially extended focal range. Nearly constant transverse resolution of approximately 1.5 microm along a focal range of 200 microm is experimentally verified with a maximum sensitivity of 105 dB. A broad-bandwidth Ti:sapphire laser allowed for an axial resolution of 3 microm in air.

Ultrahigh resolution Fourier domain optical coherence tomography.

We present, for the first time, in vivo ultrahigh resolution (~2.5 microm in tissue), high speed (10000 A-scans/second equivalent acquisition rate sustained over 160 A-scans) retinal imaging obtained with Fourier domain (FD) OCT employing a commercially available, compact (500x260mm), broad bandwidth (120 nm at full-width-at-half-maximum centered at 800 nm) Titanium:sapphire laser (Femtosource Integral OCT, Femtolasers Produktions GmbH). Resolution and sampling requirements, dispersion compensation as well as dynamic range for ultrahigh resolution FD OCT are carefully analyzed. In vivo OCT sensitivity performance achieved by ultrahigh resolution FD OCT was similar to that of ultrahigh resolution time domain OCT, although employing only 2-3 times less optical power (~300 microW). Visualization of intra-retinal layers, especially the inner and outer segment of the photoreceptor layer, obtained by FDOCT was comparable to that, accomplished by ultrahigh resolution time domain OCT, despite an at least 40 times higher data acquisition speed of FD OCT.

Performance of fourier domain vs. time domain optical coherence tomography.

In this article we present a detailed discussion of noise sources in Fourier Domain Optical Coherence Tomography (FDOCT) setups. The performance of FDOCT with charge coupled device (CCD) cameras is compared to current standard time domain OCT systems. We describe how to measure sensitivity in the case of FDOCT and confirm the theoretically obtained values. It is shown that FDOCT systems have a large sensitivity advantage and allow for sensitivities well above 80dB, even in situations with low light levels and high speed detection.

[Morphological investigations of turkey hearts after feeding ofFusarium toxins]. / Morphologische Untersuchungen von Putenherzen nach Aufnahme von Fusarientoxinen durch das Futter.

100 turkey poults (1 day of age) were housed in 16 boxes, each containing 6-7 animals. In each case 4 boxes constituted 4 feeding groups. One was the control group and the other three received a feed containing different quantities of the mycotoxin moniliformin (0.8; 1.6; 2.4 mg/kg) and beauvericin (0.8; 1.7; 2.5 mg/kg). The animals were fed for 12 weeks and then slaughtered. Pieces of the heart were examined by routine histology. The microscopical evaluation showed cell infiltration into the heart muscle and alterations of the heart muscle. However, a relation could be detected between the mycotoxin concentration in the feed and the frequency and the quality of heart alterations.

[Impact of fusarium toxins on growth and slaughter performance of broilers and turkeys]. / Einfluss von Fusarientoxinen auf die Mast- und Schlachtleistung von Broilern und Puten.

In two broiler and two turkey trials the influence of Fusarium toxins in maize on growth and slaughter performance, on residues of toxins in carcass and litters and blood parameters were investigated. In one broiler and turkey trial with naturally contaminated maize the main contaminants were deoxynivalenol (DON), moniliformin (MON and beauvericin (BEA). In one further broiler and turkey trial with inoculated maize (Fusarium subglutinans) the main contaminants were MON and BEA. The level of contamination of mycotoxins in the diets was free, low, medium and high. For every broiler trial 180 and for the turkey trials 60 and 100 one day old chicken were used. The result of these investigations shows that broiler and turkeys are not very sensitive to Fusarium toxins. Growth and slaughter performance and also blood parameters were not negative influenced by higher mycotoxin dosages in the diets.

Full range complex spectral optical coherence tomography technique in eye imaging.

We demonstrate a new implementation of complex spectral optical coherence tomography (OCT) in biomedical imaging. By reconstruction of both amplitude and phase we are able to use the negative and positive optical path differences to get images of objects of considerable thickness. An accompanying reduction of coherent noise improves the quality of the images. The property of the complex spectral OCT that permits the measurement range to be increased and permits the simultaneous use of phase and amplitude in spectral systems was not described previously. To show the potential of this technique we measured an anterior chamber of a porcine eye in vitro.

Quantitative differential phase measurement and imaging in transparent and turbid media by optical coherence tomography.

Differential phase-contrast optical coherence tomography allows one to measure the path-length differences of two transversally separated beams in the nanometer range. We calculate these path-length differences from the phase functions of the interferometric signals. Pure phase objects consisting of chromium layers containing steps of approximately 100-200-nm height were imaged. Phase differences can be measured with a precision of +/-2 degrees , corresponding to a path-difference resolution of 2-3 nm. To investigate the influence of scattering, we imaged the phase objects through scattering layers with increasing scattering coefficients. The limit of phase imaging through these layers was at approximately 8-9 mean free path lengths thick (single pass).

Differential phase measurements in low-coherence interferometry without 2pi ambiguity.

Quantitative phase measurements by low-coherence interferometry and optical coherence tomography are restricted by the well-known 2pi ambiguity to path-length differences smaller than lambda/2 . We present a method that overcomes this ambiguity. Introducing a slight dispersion imbalance between reference and sample arms of the interferometer causes the short and long wavelengths of the source spectrum to separate within the interferometric signal. This causes the phase slope to vary within the signal. The phase-difference function between two adjacent sample beam components is calculated by subtraction of their phase functions obtained from phase-sensitive interferometric signal recording. Because of the dispersive effect, the phase difference varies across the interferometric signal. The slope of that phase difference is proportional to the optical path difference, without 2pi ambiguity.

Spectral measurement of absorption by spectroscopic frequency-domain optical coherence tomography.

A new method of measurement that essentially combines Fourier-domain optical coherence tomography with spectroscopy is introduced. By use of a windowed Fourier transform it is possible to obtain, in addition to the object structure, spectroscopic information such as the absorption properties of materials. The feasibility of this new method for performing depth-resolved spectroscopy is demonstrated with a glass filter plate. The results are compared with theoretically calculated spectra by use of the well-known spectral characteristics of the light source and the filter plate.