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.

Behavioral dynamics of neuroprotective macrophage polarization in neuropathic pain observed by GHz femtosecond laser two-photon excitation microscopy.

Macrophage polarization in neurotoxic (M1) or neuroprotective (M2) phenotypes is known to play a significant role in neuropathic pain, but its behavioral dynamics and underlying mechanism remain largely unknown. Two-photon excitation microscopy (2PEM) is a promising functional imaging tool for investigating the mechanism of cellular behavior, as using near-infrared excitation wavelengths is less subjected to light scattering. However, the higher-order photobleaching effect in 2PEM can seriously hamper its applications to long-term live-cell studies. Here, we demonstrate a GHz femtosecond (fs) 2PEM that enables hours-long live-cell imaging of macrophage behavior with reduced higher-order photobleaching effect-by leveraging the repetition rate of fs pulses according to the fluorescence lifetime of fluorophores. Using this new functional 2PEM platform, we measure the polarization characteristics of macrophages, especially the long-term cellular behavior in efferocytosis, unveiling the dynamic mechanism of neuroprotective macrophage polarization in neuropathic pain. These efforts can create new opportunities for understanding long-term cellular dynamic behavior in neuropathic pain, as well as other neurobiological problems, and thus dissecting the underlying complex pathogenesis.

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.

Evaluation of Swin Transformer and knowledge transfer for denoising of super-resolution structured illumination microscopy data.

BACKGROUND: Convolutional neural network (CNN)-based methods have shown excellent performance in denoising and reconstruction of super-resolved structured illumination microscopy (SR-SIM) data. Therefore, CNN-based architectures have been the focus of existing studies. However, Swin Transformer, an alternative and recently proposed deep learning-based image restoration architecture, has not been fully investigated for denoising SR-SIM images. Furthermore, it has not been fully explored how well transfer learning strategies work for denoising SR-SIM images with different noise characteristics and recorded cell structures for these different types of deep learning-based methods. Currently, the scarcity of publicly available SR-SIM datasets limits the exploration of the performance and generalization capabilities of deep learning methods. RESULTS: In this work, we present SwinT-fairSIM, a novel method based on the Swin Transformer for restoring SR-SIM images with a low signal-to-noise ratio. The experimental results show that SwinT-fairSIM outperforms previous CNN-based denoising methods. Furthermore, as a second contribution, two types of transfer learning-namely, direct transfer and fine-tuning-were benchmarked in combination with SwinT-fairSIM and CNN-based methods for denoising SR-SIM data. Direct transfer did not prove to be a viable strategy, but fine-tuning produced results comparable to conventional training from scratch while saving computational time and potentially reducing the amount of training data required. As a third contribution, we publish four datasets of raw SIM images and already reconstructed SR-SIM images. These datasets cover two different types of cell structures, tubulin filaments and vesicle structures. Different noise levels are available for the tubulin filaments. CONCLUSION: The SwinT-fairSIM method is well suited for denoising SR-SIM images. By fine-tuning, already trained models can be easily adapted to different noise characteristics and cell structures. Furthermore, the provided datasets are structured in a way that the research community can readily use them for research on denoising, super-resolution, and transfer learning strategies.

Grazing incidence to total internal reflection fluorescence structured illumination microscopy enabled by a prism telescope.

In super-resolution structured illumination microscopy (SR-SIM) the separation between opposing laser spots in the back focal plane of the objective lens affects the pattern periodicity, and, thus, the resulting spatial resolution. Here, we introduce a novel hexagonal prism telescope which allows us to seamlessly change the separation between parallel laser beams for 3 pairs of beams, simultaneously. Each end of the prism telescope is composed of 6 Littrow prisms, which are custom-ground so they can be grouped together in the form of a tight hexagon. By changing the distance between the hexagons, the beam separation can be adjusted. This allows us to easily control the position of opposing laser spots in the back focal plane and seamlessly adjust the spatial frequency of the resulting interference pattern. This also enables the seamless transition from 2D-SIM to total internal reflection fluorescence (TIRF) excitation using objective lenses with a high numerical aperture. In linear SR-SIM the highest spatial resolution can be achieved for extreme TIRF angles. The prism telescope allows us to investigate how the spatial resolution and contrast depend on the angle of incidence near, at, and beyond the critical angle. We demonstrate this by imaging the cytoskeleton and plasma membrane of liver sinusoidal endothelial cells, which have a characteristic morphology consisting of thousands of small, transcellular pores that can only be observed by super-resolution microscopy.

High-speed TIRF and 2D super-resolution structured illumination microscopy with a large field of view based on fiber optic components.

Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fast, and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high, and the physical size of the instruments based on the implementation of their optics is still rather large. Here, we present our latest results towards realizing a new generation of compact, cost-efficient, and high-speed SR-SIM instruments. Tight integration of the fiber-based structured illumination microscope capable of multi-color 2D- and TIRF-SIM imaging, allows us to demonstrate SR-SIM with a field of view of up to 150 × 150 µm2 and imaging rates of up to 44 Hz while maintaining highest spatiotemporal resolution of less than 100 nm. We discuss the overall integration of optics, electronics, and software that allowed us to achieve this, and then present the fiberSIM imaging capabilities by visualizing the intracellular structure of rat liver sinusoidal endothelial cells, in particular by resolving the structure of their trans-cellular nanopores called fenestrations.

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse.

The availability of cost-effective, highly portable, and easy to use high-resolution live-cell imaging systems could present a significant technological break-through in challenging environments, such as high-level biosafety laboratories or sites where new viral outbreaks are suspected. We describe and demonstrate a cost-effective high-speed fluorescence microscope enabling the live tracking of virus particles across virological synapses that form between infected and uninfected T cells. The dynamics of HIV-1 proteins studied at the cellular level and the formation of virological synapses in living T cells reveals mechanisms by which cell-cell interactions facilitate infection between immune cells. Dual-color 3D fluorescence deconvolution microscopy of HIV-1 particles at frames rates of 100 frames per second allows us to follow the transfer of HIV-1 particles across the T cell virological synapse between living T cells. We also confirm the successful transfer of virus by imaging T cell samples fixed at specific time points during cell-cell virus transfer by super-resolution structured illumination microscopy.

Pseudomonas aeruginosa Clusters Toxic Nickel Nanoparticles to Enhance Survival.

Microorganisms forming a biofilm might become multidrug-resistant by information exchange. Multi-resistant, biofilm-producing microorganisms are responsible for a major portion of hospital-acquired infections. Additionally, these microorganisms cause considerable damage in the industrial sector. Here, we screened several nanoparticles of transition metals for their antibacterial properties. The nanoparticles sizes of nickel (<300 nm) and nickel oxide (<50 nm) were analyzed with transmission electron microscopy. We could show that the antibacterial efficacy of nickel and nickel oxide nanoparticles on Pseudomonas aeruginosa isolated from household appliances and Staphylococcus aureus was the highest. Interestingly, only P. aeruginosa was able to survive at high concentrations (up to 50 mM) due to clustering toxic nanoparticles out of the medium by biofilm formation. This clustering served to make the medium nearly free of nanoparticles, allowing the bacteria to continue living without contact to the stressor. We observed these clusters by CLSM, SEM, and light microscopy. Moreover, we calculated the volume of NiO particles in the bacterial biofilms based on an estimated thickness of 5 nm from the TEM images as an average volume of 3.5 × 10−6 µm3. These results give us a new perspective on bacterial defense mechanisms and might be useful in industries such as water purification.

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.

Dual color DMD-SIM by temperature-controlled laser wavelength matching.

Structured illumination microscopy (SIM) is a fast and gentle super-resolution fluorescence imaging technique, featuring live-cell compatible excitation light levels and high imaging speeds. To achieve SIM, spatial modulation of the fluorescence excitation light is employed. This is typically achieved by interfering coherent laser beams in the sample plane, which are often created by spatial light modulators (SLMs). Digital micromirror devices (DMDs) are a form of SLMs with certain advantages, such as high speed, low cost and wide availability, which present certain hurdles in their implementation, mainly the blazed grating effect caused by the jagged surface structure of the tilted mirrors. Recent works have studied this effect through modelling, simulations and experiments, and laid out possible implementations of multi-color SIM imaging based on DMDs. Here, we present an implementation of a dual-color DMD based SIM microscope using temperature-controlled wavelength matching. By carefully controlling the output wavelength of a diode laser by temperature, we can tune two laser wavelengths in such a way that no opto-mechanical realignment of the SIM setup is necessary when switching between both wavelengths. This reduces system complexity and increases imaging speed. With measurements on nano-bead reference samples, as well as the actin skeleton and membrane of fixed U2OS cells, we demonstrate the capabilities of the setup.

Endocytic Motif on a Biotin-Tagged HIV-1 Env Modulates the Co-Transfer of Env and Gag during Cell-to-Cell Transmission.

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell-cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell-cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.

Highly compact and cost-effective 2-beam super-resolution structured illumination microscope based on all-fiber optic components.

Current super-resolution structured illumination microscopes (SR-SIM) utilize relatively expensive electro-optic components and free-space optics, resulting in large setups. Moreover, high power laser sources are required to compensate for the losses associated with generating the illumination pattern by diffractive optics. Here, we present a highly compact and flexible 2D SR-SIM microscope based on all-fiber optic components (fiberSIM). Fiber-splitters deliver the laser light to the sample resulting in the interference illumination pattern. A microelectromechanical systems (MEMS) based fiber switch performs rapid pattern rotation. The pattern phase shift is achieved by the spatial displacement of one arm of the fiber interferometer using a piezoelectric crystal. Compared with existing methods, fiberSIM is highly compact and significantly reduces the SR-SIM component cost while achieving comparable results, thus providing a route to making SR-SIM technology accessible to even more laboratories in the life sciences.

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)'.

Simulating digital micromirror devices for patterning coherent excitation light in structured illumination microscopy.

Digital micromirror devices (DMDs) are spatial light modulators that employ the electro-mechanical movement of miniaturized mirrors to steer and thus modulate the light reflected off a mirror array. Their wide availability, low cost and high speed make them a popular choice both in consumer electronics such as video projectors, and scientific applications such as microscopy. High-end fluorescence microscopy systems typically employ laser light sources, which by their nature provide coherent excitation light. In super-resolution microscopy applications that use light modulation, most notably structured illumination microscopy (SIM), the coherent nature of the excitation light becomes a requirement to achieve optimal interference pattern contrast. The universal combination of DMDs and coherent light sources, especially when working with multiple different wavelengths, is unfortunately not straight forward. The substructure of the tilted micromirror array gives rise to a blazed grating, which has to be understood and which must be taken into account when designing a DMD-based illumination system. Here, we present a set of simulation frameworks that explore the use of DMDs in conjunction with coherent light sources, motivated by their application in SIM, but which are generalizable to other light patterning applications. This framework provides all the tools to explore and compute DMD-based diffraction effects and to simulate possible system alignment configurations computationally, which simplifies the system design process and provides guidance for setting up DMD-based microscopes. This article is part of the Theo Murphy meeting '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.

A Replication-Competent HIV Clone Carrying GFP-Env Reveals Rapid Env Recycling at the HIV-1 T Cell Virological Synapse.

HIV-1 infection is enhanced by cell-cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS.

Photonic technologies for liquid biopsies: recent advances and open research challenges.

The recent development of sophisticated techniques capable of detecting extremely low concentrations of circulating tumor biomarkers in accessible body fluids, such as blood or urine, could contribute to a paradigm shift in cancer diagnosis and treatment. By applying such techniques, clinicians can carry out liquid biopsies, providing information on tumor presence, evolution, and response to therapy. The implementation of biosensing platforms for liquid biopsies is particularly complex because this application domain demands high selectivity/specificity and challenging limit-of-detection (LoD) values. The interest in photonics as an enabling technology for liquid biopsies is growing owing to the well-known advantages of photonic biosensors over competing technologies in terms of compactness, immunity to external disturbance, and ultra-high spatial resolution. Some encouraging experimental results in the field of photonic devices and systems for liquid biopsy have already been achieved by using fluorescent labels and label-free techniques and by exploiting super-resolution microscopy, surface plasmon resonance, surface-enhanced Raman scattering, and whispering gallery mode resonators. This paper critically reviews the current state-of-the-art, starting from the requirements imposed by the detection of the most common circulating biomarkers. Open research challenges are considered together with competing technologies, and the most promising paths of improvement are discussed for future applications.