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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.

Early detection of retinal molecular biomarkers is crucial for addressing the unmet clinical need to prevent irreversible neural tissue damage in ophthalmic and neurodegenerative diseases. Among emerging molecular sensing techniques, non-resonant Raman spectroscopy stands out as a naturally label-free and noninvasive method, offering rich biochemical information. However, in vivo detection of non-resonant Raman spectra from retinal tissue has proven to be challenging so far. Previous studies have reported conflicting results, likely due to overwhelming pigment autofluorescence. In this study, we identified the optic nerve head as the optimal retinal location for acquiring non-resonant Raman spectra in the molecular fingerprint region. Through longitudinal intra-subject measurements, we revealed dynamic changes in the molecular composition. Furthermore, a comparative study across age groups enabled the identification of molecular alterations associated with aging. These findings establish a critical foundation for utilizing non-resonant Raman spectroscopy as an early diagnostic tool for the detection of molecular biomarkers associated with ophthalmic and neurodegenerative diseases. In vivo non-resonant Raman spectroscopy of the optic nerve head enables label-free molecular fingerprinting of the human retina, revealing longitudinal and age-related changes toward early diagnosis of ophthalmic and neurodegenerative diseases.

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

Raman sensing and microscopy are among the most specific optical technologies to identify the chemical compounds of unknown samples, and to enable label-free biomedical imaging. Here we present a method for stimulated Raman scattering spectroscopy and imaging with a time-encoded (TICO) Raman concept. We use continuous wave, rapidly wavelength-swept probe lasers and combine them with a short-duty-cycle actively modulated pump laser. Hence, we achieve high stimulated Raman gain signal levels, while still benefitting from the narrow linewidth and low noise of continuous wave operation. Our all-fibre TICO-Raman setup uses a Fourier domain mode-locked laser source to achieve a unique combination of high speed, broad spectral coverage (750–3,150 cm −1 ) and high resolution (0.5 cm −1 ). The Raman information is directly encoded and acquired in time. We demonstrate quantitative chemical analysis of a solvent mixture and hyperspectral Raman microscopy with molecular contrast of plant cells. Raman microscopes suffer from the compromise between speed and spectral information and are often unsuited for fibre beam delivery. Karpf et al. overcome these limitations using continuous-wave rapidly wavelength-swept probe lasers and a short-duty-cycle actively modulated pump laser in an all-fibre setup.

PurposeTo 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.MethodsA 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.Results3D-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.ConclusionsDifferent 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.

Raman scattering, an inelastic scattering mechanism, provides information about molecular excitation energies and can be used to identify chemical compounds. Albeit being a powerful analysis tool, especially for label-free biomedical imaging with molecular contrast, it suffers from inherently low signal levels. This practical limitation can be overcome by non-linear enhancement techniques like stimulated Raman scattering (SRS). In SRS, an additional light source stimulates the Raman scattering process. This can lead to orders of magnitude increase in signal levels and hence faster acquisition in biomedical imaging. However, achieving a broad spectral coverage in SRS is technically challenging and the signal is no longer background-free, as either stimulated Raman gain (SRG) or loss (SRL) is measured, turning a sensitivity limit into a dynamic range limit. Thus, the signal has to be isolated from the laser background light, requiring elaborate methods for minimizing detection noise. Here we analyze the detection sensitivity of a shot-noise limited broadband stimulated time-encoded Raman (TICO-Raman) system in detail. In time-encoded Raman, a wavelength-swept Fourier Domain Mode Locked (FDML) laser covers a broad range of Raman transition energies while allowing a dual-balanced detection for lowering the detection noise to the fundamental shot-noise limit.

Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. We present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.

We present an entirely fiber based laser source for non-linear imaging with a novel approach for multi-color excitation. The high power output of an actively modulated and amplified picosecond fiber laser at 1064 nm is shifted to longer wavelengths by a combination of four-wave mixing and stimulated Raman scattering. By combining different fiber types and lengths, we control the non-linear wavelength conversion in the delivery fiber itself and can switch between 1064 nm, 1122 nm, and 1186 nm on-the-fly by tuning the pump power of the fiber amplifier and modulate the seed diodes. This is a promising way to enhance the applicability of short pulsed laser diodes for bio-molecular non-linear imaging by reducing the spectral limitations of such sources. In comparison to our previous work [1, 2], we show for the first time two-photon imaging with the shifted wavelengths and we demonstrate pulse-to-pulse switching between the different wavelengths without changing the configuration.

Two-Photon Microscopy (TPM) can provide three-dimensional morphological and functional contrast in vivo. Through proper staining, TPM can be utilized to create virtual, H&E equivalent images and thus can improve throughput in histology-based applications. We previously reported on a new light source for TPM that employs a compact and robust fiber-amplified, directly modulated laser. This laser is pulse-to-pulse wavelength switchable between 1064 nm, 1122 nm, and 1186 nm with an adjustable pulse duration from 50ps to 5ns and arbitrary repetition rates up to 1MHz at kW-peak powers. Despite the longer pulse duration, it can achieve similar average signal levels compared to fs-setups by lowering the repetition rate to achieve similar cw and peak power levels. The longer pulses lead to a larger number of photons per pulse, which yields single shot fluorescence lifetime measurements (FLIM) by applying a fast 4 GSamples/s digitizer. In the previous setup, the wavelengths were limited to 1064 nm and longer. Here, we use four wave mixing in a non-linear photonic crystal fiber to expand the wavelength range down to 940 nm. This wavelength is highly suitable for imaging green fluorescent proteins in neurosciences and stains such as acridine orange (AO), eosin yellow (EY) and sulforhodamine 101 (SR101) used for histology applications. In a more compact setup, we also show virtual H&E histological imaging using a direct 1030 nm fiber MOPA.

Raman sensing and Raman microscopy are amongst the most specific optical technologies to identify the chemical compounds of unknown samples, and to enable label-free biomedical imaging with molecular contrast. However, the high cost and complexity, low speed, and incomplete spectral information provided by current technology are major challenges preventing more widespread application of Raman systems. To overcome these limitations, we developed a new method for stimulated Raman spectroscopy and Raman imaging using continuous wave (CW), rapidly wavelength swept lasers. Our all-fiber, time-encoded Raman (TICO-Raman) setup uses a Fourier Domain Mode Locked (FDML) laser source to achieve a unique combination of high speed, broad spectral coverage (750 cm-1 - 3150 cm-1) and high resolution (0.5 cm-1). The Raman information is directly encoded and acquired in time. We demonstrate quantitative chemical analysis of a solvent mixture and hyperspectral Raman microscopy with molecular contrast of plant cells.