Hepatic Vasculopathy and Regenerative Responses of the Liver in Fatal Cases of COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 (COVID-19). It may impact the heart, brain, kidney, and liver.1 Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19.2,3 Histologic evaluation has been limited to small numbers of COVID-19 cases with no control subjects2,4 and demonstrated largely heterogeneous patterns of pathology.2,3.

Resolution enhancement in an extended depth of field for volumetric two-photon microscopy.

The resolution enhancement over the extended depth of field (DOF) in the volumetric two-photon microscopy (TPM) is demonstrated by utilizing multiple orders of Bessel beams. Here the conventional method of switching laser modes (SLAM) in 2D is introduced to 3D, denoted as the volumetric SLAM (V-SLAM). The equivalent scanning beam in the TPM is a thin needle-like beam, which is generated from the subtraction between the needle-like 0th-order and the straw-like 1st-order Bessel beams. Compared with the 0th-order Bessel beam, the lateral resolution of the V-SLAM is increased by 28.6% and maintains over the axial depth of 56 µm. The V-SLAM performance is evaluated by employing fluorescent beads and a mouse brain slice. The V-SLAM approach provides a promising solution to improve the lateral resolutions for fast volumetric imaging on sparsely distributed samples.

Depth-resolved volumetric two-photon microscopy based on dual Airy beam scanning.

We demonstrate dual-Airy-beam-scanning-based volumetric two-photon microscopy (TPM) with depth-resolving capability. A pair of Airy beams with opposite acceleration is used as the excitation lights to sequentially illuminate the sample, and depth information can be resolved based on the deflection of the Airy beam. The depth-resolving range of the volumetric TPM is up to 32 µm. The advantages of the depth-resolved volumetric TPM are the depth-resolving capability over Bessel-beam-based TPM and less scanning times over traditional Gaussian-beam-based TPM. The depth-resolved volumetric TPM provides a promising fast imaging tool to study the dynamics in neural biology.

Behavioral similarity of dissipative solitons in an ultrafast fiber laser.

We unveil a new type of dissipative soliton behavior in a net-normal-dispersion bidirectional ultrafast fiber laser. That is, the bidirectional dissipative solitons will always reveal similar spectral and temporal characteristics through common gain and loss modulation, even if the transient instability is involved. The behavioral similarity enables us to accurately design the soliton patterns by introducing seed pulses through loss modulation. As a proof-of-concept application, the precise and flexible manipulation of multi-soliton patterns is demonstrated. These findings will shed new insights into the complex dissipative soliton dynamics and benefit the design of ultrafast lasers with desirable soliton patterns for practical applications.

Volumetric two-photon microscopy with a non-diffracting Airy beam.

We demonstrate a volumetric two-photon microscopy (TPM) using the non-diffracting Airy beam as illumination. Direct mapping of the imaging trajectory shows that the Airy beam extends the axial imaging range around six times longer than a traditional Gaussian beam does along the propagation direction, while maintaining a comparable lateral width. Benefiting from its non-diffracting nature, the TPM with Airy beam illumination is able not only to capture a volumetric image within a single frame, but also to acquire image structures behind a strongly scattered medium. The volumetric specimen is mapped layer by layer under Gaussian mode, while the three-dimensional structure is projected to a single two-dimensional image under Airy mode, leading to a significantly increased acquisition speed. The performance of the TPM is evaluated employing a phantom of agarose gel imbedding fluorescent beads as well as a mouse brain slice. Finally, we showcase the penetration ability of the developed Airy TPM by imaging through a scattering environment.

Ultra-broadband spatiotemporal sweeping device for high-speed optical imaging.

In this work, we propose and demonstrate a spatiotemporal sweeping fiber bundle for ultra-fast optical diagnoses over a multioctave wavelength span, ranging from ∼400 nm to ∼2000 nm. This all-optical spatiotemporal sweeping is realized by precisely controlling the length increment between individual fibers in the fiber bundle. Here, a 200-ps pixel delay increment specifically enables a pixel readout rate of up to 5 GHz. Depending on different configurations of the fiber bundle, either 1D or 2D spatiotemporal sweeping can be realized. Moreover, the high peak power of the short pulse in each pixel can facilitate the highly sensitive optical detection. To showcase its ultra-broadband operation capability, we here perform ultra-fast optical microscopy at three distinctive wavelengths, which are 710 nm, 1030 nm, and 1600 nm, and achieve tens of MHz line-scan rate and few-micrometers resolution for all three experiments. It is anticipated that this inertia-free spatiotemporal sweeping device with ultra-broad bandwidth, GHz pixel readout rate, and high detection sensitivity is promising for ultra-fast optical diagnosis, particularly when hyperspectral characteristics are desired.

1.7 µm wavelength tunable gain-switched fiber laser and its application to spectroscopic photoacoustic imaging.

Recently demonstrated bond-selective photoacoustic (PA) imaging has revealed the importance of 1.7 µm laser sources. In this Letter, we demonstrate a gain-switched thulium-doped fiber laser with continuous tuning from 1690 to 1765 nm by using an electrically driven acousto-optical tunable filter. Micro-joule laser pulses with a shot-to-shot intensity variation of 1.6% and a pulse duration of 150 ns are obtained. The laser source is then harnessed to implement a PA microscopy system, of which the lateral resolution is estimated to be 15.6 µm by scanning the edge of a black tape. The PA spectra of butter, rapeseed oil, and adipose tissue are measured, and they show a consistent absorption peak of around 1720 nm. Photoacoustic microscopy imaging of the adipose tissue demonstrates a high optical absorption contrast of lipids and the superiority of the laser for spectroscopic PA detection.

Self-healing highly-chirped fiber laser at 1.0 µm.

We demonstrate a MHz wavelength-swept fiber laser with diffraction-free and self-healing properties at the bio-favorable wavelength window of 1.0 µm. This ultrafast wavelength sweeping at a high chirp rate is all-optically realized through a newly-designed dispersive fiber that can provide a dispersion amount up to -1.7 ns/nm. It is 8 times larger than the standard single-mode fiber at this window and by adopting a double-pass configuration, the dispersion amount can be further increased to about -3.5 ns/nm, which is 23 times larger than what has previously been demonstrated. Its beam profile, a 2D Airy function, shows no obvious diffraction within a propagation distance of 2 meters and furthermore, the self-healing property is also verified by blocking the main lobe of the laser beam. This is the first wavelength-swept fiber laser equipped with diffraction-free and self-healing properties at the bio-favorable window. We believe that such effort can enable real-time data processing and a deeper penetration for the high-speed spectroscopic applications in the turbid environment.

Polystyrene microbeads influence lipid storage distribution in C. elegans as revealed by coherent anti-Stokes Raman scattering (CARS) microscopy.

The exposure of Caenorhabditis elegans to polystyrene (PS) beads of a wide range of sizes impedes feeding, by reducing food consumption, and has been linked to inhibitory effects on the reproductive capacity of this nematode, as determined in standardized toxicity tests. Lipid storage provides energy for longevity, growth, and reproduction and may influence the organismal response to stress, including the food deprivation resulting from microplastics exposure. However, the effects of microplastics on energy storage have not been investigated in detail. In this study, C. elegans was exposed to ingestible sizes of PS beads in a standardized toxicity test (96 h) and in a multigeneration test (∼21 days), after which lipid storage was quantitatively analyzed in individual adults using coherent anti-Stokes Raman scattering (CARS) microscopy. The results showed that lipid storage distribution in C. elegans was altered when worms were exposed to microplastics in form of PS beads. For example, when exposed to 0.1-µm PS beads, the lipid droplet count was 93% higher, the droplets were up to 56% larger, and the area of the nematode body covered by lipids was up to 79% higher than in unexposed nematodes. The measured values tended to increase as PS bead sizes decreased. Cultivating the nematodes for 96 h under restricted food conditions in the absence of beads reproduced the altered lipid storage and suggested that it was triggered by food deprivation, including that induced by the dilutional effects of PS bead exposure. Our study demonstrates the utility of CARS microscopy to comprehensively image the smaller microplastics (<10 µm) ingested by nematodes and possibly other biota in investigations of the effects at the level of the individual organism.

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies.

The liver as the largest organ in the human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging of the human liver is particularly challenging because of the need to cover length scales across 7 orders of magnitude (from the centimeter scale to the nanometer scale) in order to fully assess the ultrastructure of the entire organ down to the subcellular scale and probe its physiological function. This task becomes even more challenging the deeper within the organ one hopes to image, because of the strong absorption and scattering of visible light by the liver. Here, we demonstrate how optical imaging methods utilizing highly specific fluorescent labels, as well as label-free optical methods can seamlessly cover this entire size range in excised, fixed human liver tissue and we exemplify this by reconstructing the biliary tree in three-dimensional space. Imaging of tissue beyond approximately 0.5 mm length requires optical clearing of the human liver. We present the successful use of optical projection tomography and light-sheet fluorescence microscopy to derive information about the liver architecture on the millimeter scale. The intermediate size range is covered using label-free structural and chemically sensitive methods, such as second harmonic generation and coherent anti-Stokes Raman scattering microscopy. Laser-scanning confocal microscopy extends the resolution to the nanoscale, allowing us to ultimately image individual liver sinusoidal endothelial cells and their fenestrations by super-resolution structured illumination microscopy. This allowed us to visualize the human hepatobiliary system in 3D down to the cellular level, which indicates that reticular biliary networks communicate with portal bile ducts via single or a few ductuli. Non-linear optical microscopy enabled us to identify fibrotic regions extending from the portal field to the parenchyma, along with microvesicular steatosis in liver biopsies from an older patient. Lastly, super-resolution microscopy allowed us to visualize and determine the size distribution of fenestrations in human liver sinusoidal endothelial cells for the first time under aqueous conditions. Thus, this proof-of-concept study allows us to demonstrate, how, in combination, these techniques open up a new chapter in liver biopsy analysis.

High-contrast, fast chemical imaging by coherent Raman scattering using a self-synchronized two-colour fibre laser.

Coherent Raman scattering (CRS) microscopy is widely recognized as a powerful tool for tackling biomedical problems based on its chemically specific label-free contrast, high spatial and spectral resolution, and high sensitivity. However, the clinical translation of CRS imaging technologies has long been hindered by traditional solid-state lasers with environmentally sensitive operations and large footprints. Ultrafast fibre lasers can potentially overcome these shortcomings but have not yet been fully exploited for CRS imaging, as previous implementations have suffered from high intensity noise, a narrow tuning range and low power, resulting in low image qualities and slow imaging speeds. Here, we present a novel high-power self-synchronized two-colour pulsed fibre laser that achieves excellent performance in terms of intensity stability (improved by 50 dB), timing jitter (24.3 fs), average power fluctuation (<0.5%), modulation depth (>20 dB) and pulse width variation (<1.8%) over an extended wavenumber range (2700-3550 cm-1). The versatility of the laser source enables, for the first time, high-contrast, fast CRS imaging without complicated noise reduction via balanced detection schemes. These capabilities are demonstrated in this work by imaging a wide range of species such as living human cells and mouse arterial tissues and performing multimodal nonlinear imaging of mouse tail, kidney and brain tissue sections by utilizing second-harmonic generation and two-photon excited fluorescence, which provides multiple optical contrast mechanisms simultaneously and maximizes the gathered information content for biological visualization and medical diagnosis. This work also establishes a general scenario for remodelling existing lasers into synchronized two-colour lasers and thus promotes a wider popularization and application of CRS imaging technologies.

An in vitro pressure model towards studying the response of primary retinal ganglion cells to elevated hydrostatic pressures.

Glaucoma is a leading cause of blindness characterized by progressive degeneration of retinal ganglion cells (RGCs). A well-established risk factor for the development and progression of glaucoma is elevation of intraocular pressure (IOP). However, how elevated IOP leads to RGC degeneration remains poorly understood. Here, we fabricate a facile, tunable hydrostatic pressure platform to study the effect of increased hydrostatic pressure on RGC axon and total neurite length, cell body area, dendritic branching, and cell survival. The hydrostatic pressure can be adjusted by varying the height of a liquid reservoir attached to a three-dimensional (3D)-printed adapter. The proposed platform enables long-term monitoring of primary RGCs in response to various pressure levels. Our results showed pressure-dependent changes in the axon length, and the total neurite length. The proportion of RGCs with neurite extensions significantly decreased by an average of 38 ± 2% (mean ± SEM) at pressures 30 mmHg and above (p < 0.05). The axon length and total neurite length decreased at a rate of 1.65 ± 0.18 µm and 4.07 ± 0.34 µm, respectively (p < 0.001), for each mmHg increase in pressure after 72 hours pressure treatment. Dendritic branching increased by 0.20 ± 0.05 intersections/day at pressures below 25 mmHg, and decreased by 0.07 ± 0.01 intersections/day at pressures above 25 mmHg (p < 0.001). There were no significant changes in cell body area under different levels of hydrostatic pressure (p ≥ 0.05). Application of this model will facilitate studies on the biophysical mechanisms that contribute to the pathophysiology of glaucoma and provide a channel for the screening of potential pharmacological agents for neuroprotection.

Compact fs ytterbium fiber laser at 1010 nm for biomedical applications.

Ytterbium-doped fiber lasers (YDFLs) working in the near-infrared (NIR) spectral window and capable of high-power operation are popular in recent years. They have been broadly used in a variety of scientific and industrial research areas, including light bullet generation, optical frequency comb formation, materials fabrication, free-space laser communication, and biomedical diagnostics as well. The growing interest in YDFLs has also been cultivated for the generation of high-power femtosecond (fs) pulses. Unfortunately, the operating wavelengths of fs YDFLs have mostly been confined to two spectral bands, i.e., 970-980 nm through the three-level energy transition and 1030-1100 nm through the quasi three-level energy transition, leading to a spectral gap (990-1020 nm) in between, which is attributed to an intrinsically weak gain in this wavelength range. Here we demonstrate a high-power mode-locked fs YDFL operating at 1010 nm, which is accomplished in a compact and cost-effective package. It exhibits superior performance in terms of both short-term and long-term stability, i.e., <0.3% (peak intensity over 2.4 µs) and <4.0% (average power over 24 hours), respectively. To illustrate the practical applications, it is subsequently employed as a versatile fs laser for high-quality nonlinear imaging of biological samples, including two-photon excited fluorescence microscopy of mouse kidney and brain sections, as well as polarization-sensitive second-harmonic generation microscopy of potato starch granules and mouse tail muscle. It is anticipated that these efforts will largely extend the capability of fs YDFLs which is continuously tunable over 970-1100 nm wavelength range for wideband hyperspectral operations, serving as a promising complement to the gold-standard Ti:sapphire fs lasers.

Ultrafast time-stretch imaging at 932 nm through a new highly-dispersive fiber.

Optical glass fiber has played a key role in the development of modern optical communication and attracted the biotechnology researcher's great attention because of its properties, such as the wide bandwidth, low attenuation and superior flexibility. For ultrafast optical imaging, particularly, it has been utilized to perform MHz time-stretch imaging with diffraction-limited resolutions, which is also known as serial time-encoded amplified microscopy (STEAM). Unfortunately, time-stretch imaging with dispersive fibers has so far mostly been demonstrated at the optical communication window of 1.5 µm due to lack of efficient dispersive optical fibers operating at the shorter wavelengths, particularly at the bio-favorable window, i.e., <1.0 µm. Through fiber-optic engineering, here we demonstrate a 7.6-MHz dual-color time-stretch optical imaging at bio-favorable wavelengths of 932 nm and 466 nm. The sensitivity at such a high speed is experimentally identified in a slow data-streaming manner. To the best of our knowledge, this is the first time that all-optical time-stretch imaging at ultrahigh speed, high sensitivity and high chirping rate (>1 ns/nm) has been demonstrated at a bio-favorable wavelength window through fiber-optic engineering.