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.

Abstractμ-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.

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 PO43− 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.

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 twophoton 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.

Surface-enhanced Raman scattering spectroscopy (SERS) was employed to investigate the formation of self-assembled monolayers (SAMs) of biphenylthiol, 4'-nitro-1,1'-biphenyl-4-thiol, and p-terphenylthiol on Au surfaces and their structural transformations into carbon nanomembranes (CNMs) induced by electron irradiation. The high sensitivity of SERS allows us to identify two types of Raman scattering in electron-irradiated SAMs: (1) Raman-active sites exhibit similar bands as those of pristine SAMs in the fingerprint spectral region, but with indications of an amorphization process and (2) Raman-inactive sites show almost no Raman-scattering signals, except a very weak and broad D band, indicating a lack of structural order but for the presence of graphitic domains. Statistical analysis showed that the ratio of the number of Raman-active sites to the total number of measurement sites decreases exponentially with increasing the electron irradiation dose. The maximum degree of cross-linking ranged from 97 to 99% for the three SAMs. Proof-of-concept experiments were conducted to demonstrate potential applications of Raman-inactive CNMs as a supporting membrane for Raman analysis.

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 highpower 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 longterm 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 twophoton 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.

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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.