Four-wave mixing seeded by a rapid wavelength-sweeping FDML laser for nonlinear imaging at 900 nm and 1300 nm.

Four-wave mixing (FWM) enables the generation and amplification of light in spectral regions where suitable fiber gain media are unavailable. The 1300 nm and 900 nm regions are of especially high interest for time-encoded (TICO) stimulated Raman scattering microscopy and spectro-temporal laser imaging by diffracted excitation (SLIDE) two-photon microscopy. We present a new, to the best of our knowledge, FWM setup where we shift the power of a home-built fully fiber-based master oscillator power amplifier (MOPA) at 1064 nm to the 1300-nm region of a rapidly wavelength-sweeping Fourier domain mode-locked (FDML) laser in a photonic crystal fiber (PCF) creating pulses in the 900-nm region. The resulting 900-nm light can be wavelength swept over 54 nm and has up to 2.5 kW (0.2 µJ) peak power and a narrow instantaneous spectral linewidth of 70 pm. The arbitrary pulse patterns of the MOPA and the fast wavelength tuning of the FDML laser (419 kHz) allow it to rapidly tune the FWM light enabling new and faster TICO-Raman microscopy, SLIDE imaging, and other applications.

Continuous spectral zooming for in vivo live 4D-OCT with MHz A-scan rates and long coherence.

We present continuous three-dimensional spectral zooming in live 4D-OCT using a home-built FDML based OCT system with 3.28 MHz A-scan rate. Improved coherence characteristics of the FDML laser allow for imaging ranges up to 10 cm. For the axial spectral zoom feature, we switch between high resolution and long imaging range by adjusting the sweep range of our laser. We present a new imaging setup allowing for synchronized adjustments of the imaging range and lateral field of view during live OCT imaging. For this, a novel inline recalibration algorithm was implemented that enables numerical k-linearization of the raw OCT fringes for every frame instead of every volume. This is realized by acquiring recalibration data within the dead time of the raster scan at the turning points of the fast axis scanner. We demonstrate in vivo OCT images of fingers and hands at different resolution modes and show real three-dimensional zooming during live 4D-OCT. A three-dimensional spectral zooming feature for live 4D-OCT is expected to be a useful tool for a wide range of biomedical, scientific and research applications, especially in OCT guided surgery.

Imaging Inflammation - From Whole Body Imaging to Cellular Resolution.

Imaging techniques have evolved impressively lately, allowing whole new concepts like multimodal imaging, personal medicine, theranostic therapies, and molecular imaging to increase general awareness of possiblities of imaging to medicine field. Here, we have collected the selected (3D) imaging modalities and evaluated the recent findings on preclinical and clinical inflammation imaging. The focus has been on the feasibility of imaging to aid in inflammation precision medicine, and the key challenges and opportunities of the imaging modalities are presented. Some examples of the current usage in clinics/close to clinics have been brought out as an example. This review evaluates the future prospects of the imaging technologies for clinical applications in precision medicine from the pre-clinical development point of view.

Cavity length control for Fourier domain mode locked (FDML) lasers with µm precision.

In highly dispersion compensated Fourier domain mode locked (FDML) lasers, an ultra-low noise operation can only be achieved by extremely precise and stable matching of the filter tuning period and light circulation time in the cavity. We present a robust and high precision closed-loop control algorithm and an actively cavity length controlled FDML laser. The cavity length control achieves a stability of ∼0.18 mHz at a sweep repetition rate of ∼418 kHz which corresponds to a ratio of 4×10-10. Furthermore, we prove that the rapid change of the cavity length has no negative impact on the quality of optical coherence tomography using the FDML laser as light source.

Flexible A-scan rate MHz-OCT: efficient computational downscaling by coherent averaging.

In order to realize adjustable A-scan rates of fast optical coherence tomography (OCT) systems, we investigate averaging of OCT image data acquired with a MHz-OCT system based on a Fourier Domain Mode Locked (FDML) laser. Increased system sensitivity and image quality can be achieved with the same system at the cost of lower imaging speed. Effectively, the A-scan rate can be reduced in software by a freely selectable factor. We demonstrate a detailed technical layout of the strategies necessary to achieve efficient coherent averaging. Since there are many new challenges specific to coherent averaging in swept source MHz-OCT, we analyze them point by point and describe the appropriate solutions. We prove that coherent averaging is possible at MHz OCT-speed without special interferometer designs or digital phase stabilization. We find, that in our system up to ∼100x coherent averaging is possible while achieving a sensitivity increase close to the ideal values. This corresponds to a speed reduction from 3.3 MHz to 33 kHz and a sensitivity gain of 20 dB. We show an imaging comparison between coherent and magnitude averaging of a human finger knuckle joint in vivo with 121 dB sensitivity for the coherent case. Further, the benefits of computational downscaling in low sensitivity MHz-OCT systems are analyzed.

Correction: Live video rate volumetric OCT imaging of the retina with multi-MHz A-scan rates.

[This corrects the article DOI: 10.1371/journal.pone.0213144.].

Live video rate volumetric OCT imaging of the retina with multi-MHz A-scan rates.

Surgical microscopes are vital tools for ophthalmic surgeons. The recent development of an integrated OCT system for the first time allows to look at tissue features below the surface. Hence, these systems can drastically improve the quality and reduce the risk of surgical interventions. However, current commercial OCT-enhanced ophthalmic surgical microscopes provide only one additional cross sectional view to the standard microscope image and feature a low update rate. To present volumetric data at a high update rate, much faster OCT systems than the ones applied in today's surgical microscopes need to be developed. We demonstrate live volumetric retinal OCT imaging, which may provide a sufficiently large volume size (330x330x595 Voxel) and high update frequency (24.2 Hz) such that the surgeon may even purely rely on the OCT for certain surgical maneuvers. It represents a major technological step towards the possible application of OCT-only surgical microscopes in the future which would be much more compact thus enabling many additional minimal invasive applications. We show that multi-MHz A-scan rates are essential for such a device. Additionally, advanced phase-based OCT techniques require 3D OCT volumes to be detected with a stable optical phase. These techniques can provide additional functional information of the retina. Up to now, classical OCT was to slow for this, so our system can pave the way to holographic OCT with a traditional confocal flying spot approach. For the first time, we present point scanning volumetric OCT imaging of the posterior eye with up to 191.2 Hz volume rate. We show that this volume rate is high enough to enable a sufficiently stable optical phase to a level, where remaining phase errors can be corrected. Applying advanced post processing concepts for numerical refocusing or computational adaptive optics should be possible in future with such a system.

High-resolution retinal swept source optical coherence tomography with an ultra-wideband Fourier-domain mode-locked laser at MHz A-scan rates.

We present a new 1060 nm Fourier domain mode locked laser (FDML laser) with a record 143 nm sweep bandwidth at 2∙ 417 kHz  =  834 kHz and 120 nm at 1.67 MHz, respectively. We show that not only the bandwidth alone, but also the shape of the spectrum is critical for the resulting axial resolution, because of the specific wavelength-dependent absorption of the vitreous. The theoretical limit of our setup lies at 5.9 µm axial resolution. In vivo MHz-OCT imaging of human retina is performed and the image quality is compared to the previous results acquired with 70 nm sweep range, as well as to existing spectral domain OCT data with 2.1 µm axial resolution from literature. We identify benefits of the higher resolution, for example the improved visualization of small blood vessels in the retina besides several others.

Wavelength agile multi-photon microscopy with a fiber amplified diode laser.

Multi-photon microscopy is a powerful tool in biomolecular research. Less complex and more cost effective excitation light sources will make this technique accessible to a broader community. Semiconductor diode seeded fiber lasers have proven to be especially robust, low cost and easy to use. However, their wavelength tuning range is often limited, so only a limited number of fluorophores can be accessed. Therefore, different approaches have been proposed to extend the spectral coverage of these lasers. Recently, we showed that four-wave mixing (FWM) assisted stimulated Raman scattering (SRS) can be harnessed to red-shift high power pulses from 1064 nm to a narrowband output at 1122 nm and 1186 nm and therefore extend the number of accessible fluorophores. In this contribution, we show the applicability of all three wavelengths for multi-photon microscopy and analyze the performance.

Combined in-depth, 3D, en face imaging of the optic disc, optic disc pits and optic disc pit maculopathy using swept-source megahertz OCT at 1050 nm.

PURPOSE: To demonstrate papillary imaging of eyes with optic disc pits (ODP) or optic disc pit associated maculopathy (ODP-M) with ultrahigh-speed swept-source optical coherence tomography (SS-OCT) at 1.68 million A-scans/s. To generate 3D-renderings of the papillary area with 3D volume-reconstructions of the ODP and highly resolved en face images from a single densely-sampled megahertz-OCT (MHz-OCT) dataset for investigation of ODP-characteristics. METHODS: A 1.68 MHz-prototype SS-MHz-OCT system at 1050 nm based on a Fourier-domain mode-locked laser was employed to acquire high-definition, 3D datasets with a dense sampling of 1600 × 1600 A-scans over a 45° field of view. Six eyes with ODPs, and two further eyes with glaucomatous alteration or without ocular pathology are presented. 3D-rendering of the deep papillary structures, virtual 3D-reconstructions of the ODPs and depth resolved isotropic en face images were generated using semiautomatic segmentation. RESULTS: 3D-rendering and en face imaging of the optic disc, ODPs and ODP associated pathologies showed a broad spectrum regarding ODP characteristics. Between individuals the shape of the ODP and the appending pathologies varied considerably. MHz-OCT en face imaging generates distinct top-view images of ODPs and ODP-M. MHz-OCT generates high resolution images of retinal pathologies associated with ODP-M and allows visualizing ODPs with depths of up to 2.7 mm. CONCLUSIONS: Different patterns of ODPs can be visualized in patients for the first time using 3D-reconstructions and co-registered high-definition en face images extracted from a single densely sampled 1050 nm megahertz-OCT (MHz-OCT) dataset. As the immediate vicinity to the SAS and the site of intrapapillary proliferation is located at the bottom of the ODP it is crucial to image the complete structure and the whole depth of ODPs. Especially in very deep pits, where non-swept-source OCT fails to reach the bottom, conventional swept-source devices and the MHz-OCT alike are feasible and beneficial methods to examine deep details of optic disc pathologies, while the MHz-OCT bears the advantage of an essentially swifter imaging process.

Pulse-to-pulse wavelength switching of a nanosecond fiber laser by four-wave mixing seeded stimulated Raman amplification.

We report on a multi-color fiber laser based on four-wave mixing (FWM) and stimulated Raman scattering (SRS), delivering rapidly wavelength switchable narrowband output at 1064, 1122, and 1186 nm. High-power pulses from a nanosecond pulsed fiber master oscillator power amplifier at 1064 nm are combined with 1122 nm of seed light for Raman amplification at the first Stokes order in a standard single-mode fiber. With increasing power, we observe a narrowband spectral component at 1186 nm, without any additional seed or resonator at this wavelength. We analyze this occurrence of a narrowband second Stokes order both experimentally and theoretically and suggest it is a result of FWM seeding of the SRS amplification in the fiber. We demonstrate that the wavelength shifting can be controlled electronically within microseconds for very rapid and even pulse-to-pulse wavelength changes. This wavelength conversion method can extend the spectral coverage of single-wavelength fiber lasers for biomedical imaging.

Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

Combined 60° Wide-Field Choroidal Thickness Maps and High-Definition En Face Vasculature Visualization Using Swept-Source Megahertz OCT at 1050 nm.

PURPOSE: To demonstrate ultrahigh-speed swept-source optical coherence tomography (SS-OCT) at 1.68 million A-scans/s for choroidal imaging in normal and diseased eyes over a ∼60° field of view. To investigate and correlate wide-field three-dimensional (3D) choroidal thickness (ChT) and vascular patterns using ChT maps and coregistered high-definition en face images extracted from a single densely sampled Megahertz-OCT (MHz-OCT) dataset. METHODS: High-definition, ∼60° wide-field 3D datasets consisting of 2088 × 1024 A-scans were acquired using a 1.68 MHz prototype SS-OCT system at 1050 nm based on a Fourier-domain mode-locked laser. Nine subjects (nine eyes) with various chorioretinal diseases or without ocular pathology are presented. Coregistered ChT maps, choroidal summation maps, and depth-resolved en face images referenced to either the retinal pigment epithelium or the choroidal-scleral interface were generated using manual segmentation. RESULTS: Wide-field ChT maps showed a large inter- and intraindividual variance in peripheral and central ChT. In only four of the nine eyes, the location with the largest ChT was coincident with the fovea. The anatomy of the large lumen vessels of the outer choroid seems to play a major role in determining the global ChT pattern. Focal ChT changes with large thickness gradients were observed in some eyes. CONCLUSIONS: Different ChT and vascular patterns could be visualized over ∼60° in patients for the first time using OCT. Due to focal ChT changes, a high density of thickness measurements may be favorable. High-definition depth-resolved en face images are complementary to cross sections and thickness maps and enhance the interpretation of different ChT patterns.

Ultra-widefield retinal MHz-OCT imaging with up to 100 degrees viewing angle.

We evaluate strategies to maximize the field of view (FOV) of in vivo retinal OCT imaging of human eyes. Three imaging modes are tested: Single volume imaging with 85° FOV as well as with 100° and stitching of five 60° images to a 100° mosaic (measured from the nodal point). We employ a MHz-OCT system based on a 1060nm Fourier domain mode locked (FDML) laser with a depth scan rate of 1.68MHz. The high speed is essential for dense isotropic sampling of the large areas. Challenges caused by the wide FOV are discussed and solutions to most issues are presented. Detailed information on the design and characterization of our sample arm optics is given. We investigate the origin of an angle dependent signal fall-off which we observe towards larger imaging angles. It is present in our 85° and 100° single volume images, but not in the mosaic. Our results suggest that 100° FOV OCT is possible with current swept source OCT technology.