Watt-level all polarization-maintaining femtosecond fiber laser source at 1100 †nm.

We demonstrate a compact watt-level all polarization-maintaining (PM) femtosecond fiber laser source at 1100 nm. The fiber laser source is seeded by an all PM fiber mode-locked laser employing a nonlinear amplifying loop mirror. The seed laser can generate stable pulses at a fundamental repetition rate of 40.71 MHz with a signal-to-noise rate of >100 dB and an integrated relative intensity noise of only ∼0.061%. After two-stage external amplification and pulse compression, an output power of ∼1.47 W (corresponding to a pulse energy of ∼36.1 nJ) and a pulse duration of ∼251 fs are obtained. The 1100 nm femtosecond fiber laser is then employed as the excitation light source for multicolor multi-photon fluorescence microscopy of Chinese hamster ovary (CHO) cells stably expressing red fluorescent proteins.

Uptake of microplastics and impacts on plant traits of savoy cabbage.

Anthropogenic influences such as plastic pollution are causing serious environmental problems. While effects of microplastics on marine organisms are well studied, less is known about effects of plastic particles on terrestrial organisms such as plants. We investigated the effects of microplastic particles on different growth and metabolic traits of savoy cabbage (Brassica oleracea var. sabauda). Sections of seedlings exposed to polystyrene particles were analysed by coherent Raman scattering microscopy. These analyses revealed an uptake of particles in a size range of 0.5 µm to 2.0 µm into cells of the hypocotyl. Furthermore, plants were grown in substrate amended with polyethylene and polystyrene particles of different sizes (s1: 200-500 µm; s2: 100-200 µm; s3: 20-100 µm; s4: < 100 µm, with most particles < 20 µm; s5: < 20 µm) and in different concentrations (c1 = 0.1%, c2 = 0.01%, c3 = 0.001%). After several weeks, shoot and root biomass were harvested. Leaves were analysed for their carbon to nitrogen ratio, while amino acid and glucosinolate composition were measured using high performance liquid chromatography. Plastic type, particle size and concentration showed distinct effects on certain plant traits. Shoot biomass was interactively influenced by size and concentration of polyethylene, while root biomass was not modified by any of the plastic exposure treatments. Likewise, the composition and total concentrations of leaf amino acids were not affected, but the leucine concentration was significantly increased in several of the plastic-exposed plants. Glucosinolates were also slightly altered, depending on the particle size. Some of the observed effects may be independent of plastic uptake, as larger particles were not taken up but still could affect plant traits. For example, in the rhizosphere plastic particles may increase the water holding capacity of the soil, impacting some of the plant traits. In summary, this study shows how important the plastic type, particle size and concentration are for the uptake of microplastics and their effects on plant traits, which may have important implications for crops, but also for ecosystems.

Generation of a µ-1,2-hydroperoxo FeIIIFeIII and a µ-1,2-peroxo FeIVFeIII Complex.

µ-1,2-Peroxo-diferric intermediates (P) of non-heme diiron enzymes are proposed to convert upon protonation either to high-valent active species or to activated P' intermediates via hydroperoxo-diferric intermediates. Protonation of synthetic µ-1,2-peroxo model complexes occurred at the µ-oxo and not at the µ-1,2-peroxo bridge. Here we report a stable µ-1,2-peroxo complex {FeIII(µ-O)(µ-1,2-O2)FeIII} using a dinucleating ligand and study its reactivity. The reversible oxidation and protonation of the µ-1,2-peroxo-diferric complex provide µ-1,2-peroxo FeIVFeIII and µ-1,2-hydroperoxo-diferric species, respectively. Neither the oxidation nor the protonation induces a strong electrophilic reactivity. Hence, the observed intramolecular C-H hydroxylation of preorganized methyl groups of the parent µ-1,2-peroxo-diferric complex should occur via conversion to a more electrophilic high-valent species. The thorough characterization of these species provides structure-spectroscopy correlations allowing insights into the formation and reactivities of hydroperoxo intermediates in diiron enzymes and their conversion to activated P' or high-valent intermediates.

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.

Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

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.

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.

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.

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Alveolar type II (ATII) cells in the peripheral human lung spontaneously differentiate toward ATI cells, thus enabling air-blood barrier formation. Here, linear Raman and coherent anti-Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated with gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI-like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for three-dimensional visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label-free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.

Pulse length variation causing spectral distortions in OPO-based hyperspectral coherent Raman scattering microscopy.

Picosecond optical parametric oscillators (OPOs) with broad wavelength tunability are frequently used as light sources in hyperspectral coherent Raman scattering (CRS) microscopy. We investigate how changes in the pulse length during OPO wavelength tuning of the pump beam affect hyperspectral CRS imaging. We find that significant distortions of the resulting CRS spectra occur if the OPO is operated without monitoring pulse length variations. By utilizing a custom-written MATLAB based control program to counteract changes in pulse length, normalized and reproducible data sets can be acquired. We demonstrate this by comparing hyperspectral data obtained from pure substances, as well as relevant biological specimens.

Analyzing life-history traits and lipid storage using CARS microscopy for assessing effects of copper on the fitness of Caenorhabditis elegans.

Lipid storage provides energy for cell survival, growth, and reproduction and is closely related to the organismal response to stress imposed by toxic chemicals. However, the effects of toxicants on energy storage as it impacts certain life-history traits have rarely been investigated. Here, we used the nematode Caenorhabditis elegans as a test species for a chronic exposure to copper (Cu) at EC20 (0.50 mg Cu/l). Effects on the fatty acid distribution in C. elegans body were determined using coherent anti-Stokes Raman spectroscopy (CARS) to link population fitness responses with individual ecophysiological responses. Cu inhibited nematode reproductive capacity and offspring growth in addition to shortening the lifespan of exposed individuals. In adult nematodes, Cu exposure led to significant reduction of lipid storage compared to the Cu-free control: Under Cu, lipids filled only 0.5% of the nematode body volume vs. 7.5% in control nematodes, lipid droplets were on average 74% smaller and the number of tiny lipids (0-10 µm2) was increased. These results suggest that (1) Cu has an important effect on the life-history traits of nematodes; (2) the quantification of lipid storage can provide important information on the response of organisms to toxic stress; and (3) CARS microscopy is a promising tool for non-invasive quantitative and qualitative analyses of lipids as a measure of nematode fitness.

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.

Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy.

Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation.

Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

Palladium-catalyzed direct arylation of heteroaromatic compounds: improved conditions utilizing controlled microwave heating.

A versatile and rapid microwave-assisted procedure for the palladium-catalyzed direct arylation of heterocycles by aryl bromides and heteroaryl bromides is described. This novel protocol features short coupling times (10-60 min) and low catalyst loadings (1 mol %) and allows the successful arylation of previously unreactive heterocyclic substrates.

Rapid nickel-catalyzed Suzuki-Miyaura cross-couplings of aryl carbamates and sulfamates utilizing microwave heating.

High-speed and scalable nickel-catalyzed cross-coupling of arylboronic acids with aryl carbamates and sulfamates is achieved by using sealed-vessel microwave processing.

Toward the synthesis of the antibiotic branimycin: novel approaches to highly substituted cis-decalin systems.

A variety of highly functionalized cis-decalin systems have been prepared by means of the stereoselective transannular Diels-Alder (TADA) reaction of a (Z,E,Z,Z)-tetraene macrolide, and by means of intramolecular nitrile oxide olefin (INOC) or ring-closing metathesis (RCM) annulations to quinic acid derivatives.