MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation.

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F1 Fo -ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super-resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re-expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre-existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60-subcomplex is sufficient for CJ formation, whereas the Mic10-subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F1 Fo -ATP synthase, fine-tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.

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.

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.

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.

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.

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.

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.

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)'.

Smart microgels investigated by super-resolution fluorescence microscopy: influence of the monomer structure on the particle morphology.

In a recent publication [Bergmann et al. Phys. Chem. Chem. Phys., 2018, 20, 5074-5083] we presented a method which enables to investigate the morphology of microgels by superresolution fluorescence microscopy. Here, this method is applied to three microgel species, based on N-isopropylmethacrylamide (NIPMAM), N-n-propylacrylamide (NNPAM) and N-n-propylmethacrylamide (NNPMAM)) with 5, 7.5 and 10 mol% cross-linker, respectively. Super-resolution microscopy reveals differences of the network morphology of the synthesized particles showing the importance of the monomer structure.

Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies.

Endotoxin (lipooligosaccharide, LOS, and lipopolysaccharide, LPS) is the major molecular component of Gram-negative bacteria outer membrane, and very potent pro-inflammatory substance. Visualizing and tracking the distribution of the circulating endotoxin is one of the fundamental approaches to understand the molecular aspects of infection with subsequent inflammatory and immune responses, LPS also being a key player in the molecular dialogue between microbiota and host. While fluorescently labeled LPS has previously been used to track its subcellular localization and colocalization with TLR4 receptor and downstream effectors, our knowledge on lipopolysaccharide (LOS) localization and cellular activity remains almost unexplored. In this study, LOS was labeled with a novel fluorophore, Cy7N, featuring a large Stokes-shifted emission in the deep-red spectrum resulting in lower light scattering and better imaging contrast. The LOS-Cy7N chemical identity was determined by mass spectrometry, and immunoreactivity of the conjugate was evaluated. Interestingly, its application to microscopic imaging showed a faster cell internalization compared to LPS-Alexa488, despite that it is also CD14-dependent and undergoes the same endocytic pathway as LPS toward lysosomal detoxification. Our results suggest the use of the new infrared fluorophore Cy7N for cell imaging of labeled LOS by confocal fluorescence microscopy, and propose that LOS is imported in the cells by mechanisms different from those responsible for LPS uptake.

SIMPLE: Structured illumination based point localization estimator with enhanced precision.

We present a structured illumination microscopy based point localization estimator (SIMPLE) that achieves a 2-fold increase in single molecule localization precision compared to conventional centroid estimation methods. SIMPLE advances the recently introduced MINFLUX concept by using precisely phase-shifted sinusoidal wave patterns as nanometric rulers for simultaneous particle localization based on photon count variation over a 20 µm field of view. We validate SIMPLE in silico and experimentally on a TIRF-SIM setup using a digital micro-mirror device (DMD) as a spatial light modulator.

Super-Resolution Structured Illumination Microscopy.

Super-resolved structured illumination microscopy (SR-SIM) is among the most rapidly growing fluorescence microscopy techniques that can surpass the optical diffraction limit. The strength of SR-SIM is that it can be readily applied to samples prepared for conventional fluorescence microscopy, requiring no sophisticated sample preparation protocols. As an extension of wide-field fluorescence microscopy, it is inherently capable of multicolor imaging and optical sectioning and, with sufficiently fast implementations, permits live cell imaging. Image reconstruction, however, currently relies on sophisticated computational procedures, susceptible to reconstruction artifacts, requiring trained users to recognize and avoid them. Here, we review the latest developments in SR-SIM research. Starting from a historical overview of the development of SR-SIM, we review how this method can be implemented in various experimental schemes, we provide an overview of the important parameters involved in successful image reconstruction, we summarize recent biological applications, and we provide a brief outlook of the directions in which we believe SR-SIM is headed in the future.

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.

Surface-Enhanced Raman Spectroscopy of Carbon Nanomembranes from Aromatic Self-Assembled Monolayers.

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.

Super-resolution optical microscopy resolves network morphology of smart colloidal microgels.

We present a new method to resolve the network morphology of colloidal particles in an aqueous environment via super-resolution microscopy. By localization of freely diffusing fluorophores inside the particle network we can resolve the three dimensional structure of one species of colloidal particles (thermoresponsive microgels) without altering their chemical composition through copolymerization with fluorescent monomers. Our approach utilizes the interaction of the fluorescent dye rhodamine 6G with the polymer network to achieve an indirect labeling. We calculate the 3D structure from the 2D images and compare the structure to previously published models for the microgel morphology, e.g. the fuzzy sphere model. To describe the differences in the data an extension of this model is suggested. Our method enables the tailor-made fabrication of colloidal particles which are used in various applications, such as paints or cosmetics, and are promising candidates for drug delivery, smart surface coatings, and nanocatalysis. With the precise knowledge of the particle morphology an understanding of the underlying structure-property relationships for various colloidal systems is possible.

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.

Comment on "Magnetic-field-enabled resolution enhancement in super-resolution imaging" by M. Zhang et al., Phys. Chem. Chem. Phys., 2015, 17, 6722-6727.

Certain fluorophores, in particular those that can undergo photoinduced radical pair reactions are known to exhibit a magnetic field dependent fluorescence summarized in the term magnetic field effect (MFE). We tried to reproduce experiments that reported magnetic field enhanced fluorescence for commonly used organic dyes with a high quantum yield suitable for single molecule localization microscopy. We find that the enhanced fluorescence is due to fluorescence reflected by the magnet's surface rather than MFE.

Safety: off site - out of mind?.

More organizations are mov- ing to patient treatment out- side of the traditional hospital and into practices and other off-site locations. As this tran- sition occurs, it is important to establish health and safety programs for the patients and staff at these locations. This is true even if the practices are not part of a hospital accredi- tation program as there are other regulatory agencies which affect the operations of these off-site locations.