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Future Directions in Advanced Mycological Microscopy
Abstract and Figures
The field of mycology is poised to exploit the many recent advances in microscopic tools and instrumentation for cell biology. This chapter first outlines the latest developments in biosensors that will prove useful for targeting specific events in the context of single fungal cells, mycelia, or fungal-plant systems. Next, we focus on microscopic methods, in particular electron based, capable of generating three-dimensional (3D) data at single-molecule resolution. Combining the capabilities of any of the powerful microscopy platforms discussed in this volume (Chaps. 1–7) leads to correlative microscopy which vastly expands the range of image scale and data complexity. This sophisticated approach combines data collected separately or simultaneously from individual or hybrid microscopes, respectively, offering complementary internal and external spatial, structural, biochemical, and biophysical information on one sample. Finally, we review the recent select advances in imaging technology that we believe hold special promise to glean new insights from the inner working of fungal cells with unprecedented spatial and temporal resolutions.
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... 18: Principle of the laser confocal microscope[236] While an optical microscope produces a blurred image of the sample (a sample is never perfectly flat), a confocal microscope has the ability to reconstruct a sharp one. It is achieved thanks to the insertion of a pinhole between the mirror and thedetector on the focal plane of the tube lens. ...
Large-scale production of superconducting radio-frequency (SRF) cavities is an industrial challenge, not only because of the increasing number of unit for future projects but also because of requirements in term of reliability, reproducibility and performances very close to the physical limit of polycrystalline bulk Niobium. Nowadays, XFEL (DESY) and LHC (CERN) are the largest existing accelerators which are based on SRF technology. Even more challenging SRF accelerator projects like ILC (International Linear Collider) and FCC (Future Circular Collider) are being studied. For such large-scale facilities, higher performances, reduction in fabrication and operation costs are required and essential to proceed with industrialization.A pathway to reduce these costs and improve performances has been studied in this work. It consists in optimizing the cleaning process of cavity surfaces. Indeed, pollution and crystal defects on the surface created during fabrication steps of a SRF cavity have to be removed to ensure optimal superconducting performances. In order to get rid of impurities and to recover crystal structure, two polishing techniques are routinely used: the buffered chemical polishing (BCP) and electro-polishing (EP). However, these techniques involve highly concentrated acids, which lead to high operation costs and safety concerns. A way to overcome the aforementioned drawbacks and make the construction of future accelerators possible would be to replace or complement the conventional chemical polishing by alternative polishing techniques.Mechanical polishing has already been applied in SRF-community for decades by using centrifugal barrel polishing (CBP). This technique could provide a better surface roughness and could be more efficient at removing some surface defects compared to EP and BCP. However, this process does not satisfy requirements for large-scale production due to strong surface pollution and an extremely long processing time. The first part of the PhD work consisted in reproducing the state of the art, understanding its limitations and optimizing the recipe by the reduction of the surface pollution (embedded abrasives) and processing time (reduction of intermediate steps). As a conclusion to this first study, CBP could only be a complementary polishing technique to chemical treatments.The second part of the work focused on metallographic flat polishing. This technique cannot be directly applied on enclosed geometries however, it can remove efficiently surface defects (impurities and crystal damages) created during the fabrication of Niobium sheet. A 2-step process, inspired from metallographic techniques (typically 5-6 steps) has been successfully developed and optimized on Niobium for SRF applications. This process provides not only an improved roughness compared to conventional chemical treatments but also preserve the crystal quality underneath the surface, over the field penetration depth. Additional studies have to be now carried out to optimize conventional forming process or characterize alternative techniques to limit surface damages and preserve material quality as much as possible.Last but not least, the work done is of first importance for the future of SRF cavities meaning the use of new superconducting materials as thin films. The quality of thin-films of alternative superconductors depends strongly on the surface state of the substrate, typically polycrystalline bulk Niobium or Copper.
There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.
Synchrotron infrared nano-spectroscopy (SINS) provides sensitive vibrational chemical contrast with < 25 nm spatial resolution, enabling investigations of nanoscale surface phenomena that were previously impossible to study with infrared techniques.
Fungi interact with plants in various ways, with each interaction giving rise to different alterations in both partners. While fungal pathogens have detrimental effects on plant physiology, mutualistic fungi augment host defence responses to pathogens and/or improve plant nutrient uptake. Tropic growth towards plant roots or stomata, mediated by chemical and topographical signals, has been described for several fungi, with evidence of species-specific signals and sensing mechanisms. Fungal partners secrete bioactive molecules such as small peptide effectors, enzymes and secondary metabolites which facilitate colonization and contribute to both symbiotic and pathogenic relationships. There has been tremendous advancement in fungal molecular biology, omics sciences and microscopy in recent years, opening up new possibilities for the identification of key molecular mechanisms in plant-fungal interactions, the power of which is often borne out in their combination. Our fragmentary knowledge on the interactions between plants and fungi must be made whole to understand the potential of fungi in preventing plant diseases, improving plant productivity and understanding ecosystem stability. Here, we review innovative methods and the associated new insights into plant-fungal interactions.
Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1-43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.
P>We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.
Polar (directional) cell growth, a key cellular mechanism shared among a wide range of species, relies on targeted insertion of new material at specific locations of the plasma membrane. How these cell polarity sites are stably maintained during massive membrane insertion has remained elusive. Conventional live-cell optical microscopy fails to visualize polarity site formation in the crowded cell membrane environment because of its limited resolution. We have used advanced live-cell imaging techniques to directly observe the localization, assembly, and disassembly processes of cell polarity sites with high spatiotemporal resolution in a rapidly growing filamentous fungus, Aspergillus nidulans. We show that the membrane-associated polarity site marker TeaR is transported on microtubules along with secretory vesicles and forms a protein cluster at that point of the apical membrane where the plus end of the microtubule touches. There, a small patch of membrane is added through exocytosis, and the TeaR cluster gets quickly dispersed over the membrane. There is an incessant disassembly and reassembly of polarity sites at the growth zone, and each new polarity site locus is slightly offset from preceding ones. On the basis of our imaging results and computational modeling, we propose a transient polarity model that explains how cell polarity is stably maintained during highly active directional growth.
Since publication of the first edition of Volume I in 1994, the field of fungal biology has developed tremendously, mainly through the advancement of various molecular techniques and international fungal genome projects. To accommodate these developments, the second edition has been completely updated. Six chapters have been revised by former authors, others by newly recruited experts, and also novel subjects, emerged in more recent years, have been added to the book.
Leading scientists in the field have compiled comprehensive overviews as well as latest results obtained from cytological, genetic and molecular studies. Topics include: cellular and colony growth of fungi, cellular fusion and incompatibility, senescence and programmed cell death, environmental and physiological signalling in differentiation processes, asexual and sexual reproduction, mitosis and meiosis of various types of fungi. Both parallels and differences become visible between individual fungi as well as between fungal classes.
A high resolution X-ray microscope endstation was constructed on a wiggler beamline at the National Synchrotron Radiation Laboratory (NSRL). Parameters of the ellipsoidal glass capillaries as condensers were calculated and designed based on the illumination requests in the X-ray microscope system. Performance of the ellipsoidal glass capillaries was tested. The results indicate that the beam size agrees with the designed parameters and focus efficiencies of the ellipsoidal glass capillary condensers are better than 85%.
Computed Tomography gives a detailed overview of various aspects of computed tomography. It discusses X-ray CT tomography from a historical point of view, the design and physical operating principles of computed tomography apparatus, the algorithms of image reconstruction and the quality assessment criteria of tomography scanners. Algorithms of image reconstruction from projections, a crucial problem in medical imaging, are considered in depth. The author gives descriptions of the reconstruction methods related to tomography scanners with a parallel X-ray beam, trough solutions with fan-shaped beam and successive modifications of spiral scanners. Computed Tomography contains a dedicated chapter for those readers who are interested in computer simulations based on studies of reconstruction algorithms. The information included in this chapter will enable readers to create a simulation environment in which virtual tomography projections can be obtained in all basic projection systems. This monograph is a valuable study on computed tomography that will be of interest to advanced students and researchers in the fields of biomedical engineering, medical electronics, computer science and medicine.
The possibility of using two Fourier transform mid-infrared spectroscopic techniques was investigated with the purpose of rapid detection of mycotoxin-producing Fusarium fungi on wheat, as an indicator for the presence of the mycotoxin deoxynivalenol (DON). Samples of a single wheat genotype (Monika, blanks and contaminated with Fusarium graminearum) were ground and analyzed applying the diffuse reflection (DR) and attenuated total reflection (ATR) modes. The recorded spectra were evaluated with principal component analysis and the blank and contaminated samples were classified by cluster analysis. Besides, the possibility was examined of determining DON on the basis of the ratio of ATR signals at 1709 cm-1 and 1743 cm-1. Reference measurements were performed by high performance liquid chromatography with diode array detection. The concentration range for contaminated samples was 2.51-12.14 mg/kg. Classification efficiency was 100% for ATR spectra, whereas DR spectra did not show so obvious clustering of contaminated and blank samples. The ATR technique appeared advantageous owing to its easier use and interpretation of results, which were better in respect of classification and quantification. Quantification using partial least squares (PLS1) regression, as well as multiple linear regression (MLR) showed good correlation with DON reference data for the mentioned wheat genotype.
Confocal scanning optical microscopy (CSOM) is a new optical microscopic technique, which offers significant advantages over conventional microscopy. In laser scanning optical microscopy (SOM), the specimen is scanned by a diffractionlimited spot of laser light, and light transmitted or reflected by the in-focus illuminated volume element (voxel) of the specimen, or the fluorescence emission excited within it by the incident light, is focused onto a photodetector. As the illuminating spot is scanned over the specimen, the electrical output from this detector is displayed at the appropriate spatial position on a TV monitor, thus building up a two-dimensional image.
In the confocal mode, an aperture, usually slightly smaller in diameter than the Airy disc image, is positioned in the image plane in front of the detector, at a position confocal with the in-focus voxel. Light emanating from this in-focus voxel thus passes through the aperture to the detector, while that from any region above or below the focal plane is defocused at the aperture plane and is thus largely prevented from reaching the detector, contributing essentially nothing to the confocal image. It is this ability to reduce out-of-focus blur, and thus permit accurate non-invasive optical sectioning, that makes confocal scanning microscopy so well suited for the imaging and three-dimensional tomography of stained biological specimens.
In this review, I explain the principles of scanning optical microscopy and blur-free confocal imaging, discuss the various imaging modes of confocal microscopy, and illustrate some of its early applications.