Morphology visualization of irregular shape bacteria by electron holography and tomography

In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized by phase retrieval employing off‐axis electron holography (EH) and 3D reconstruction electron tomography using high‐angle annular dark field scanning transmission electron microscopy (HAADF‐STEM). Bacteria interacting with gold nanoparticles (AuNP) acquired a shrunken or irregular shape due to air dehydration processing. STEM imaging shows the attachment of AuNP on the surface of cells and suggests an irregular 3D morphology of the specimen. The phase reconstruction demonstrates that off‐axis electron holography can reveal with a single hologram the morphology of the specimen and the distribution of the functionalized AuNPs. In addition, EH reduces significantly the acquisition time and the cumulative radiation damage (in three orders of magnitude) over biological samples in comparison with multiple tilted electron expositions intrinsic to electron tomography, as well as the processing time and the reconstruction artifacts that may arise during tomogram reconstruction.     

3D elemental and structural analysis of biological specimens using electrons and ions

We demonstrate the utility of focused ion beam scanning electron microscopy combined with energy dispersive x-ray spectrometry for 3D morphological and elemental correlative analysis of subcellular features. Although recent advances in super-resolution light microscopy techniques and traditional transmission electron microscopy methods can provide cellular imaging at a wide range of length scales, simultaneous 3D morphological and elemental imaging of cellular features at nanometre scale can only be achieved with techniques such as focused ion beam scanning electron microscopy with energy dispersive x-ray spectrometry capability. We demonstrate the technique by analysing the 3D silicon cell wall structure of a marine diatom, Thalassiosira pseudonana. This study also highlights the limitations of the technique in its current state and suggests several possible improvements needed for the routine use of the technique for biological specimens.     

Study of hyperpigmentation in human skin disorder using different electron microscopy techniques

There is a global trend of increase in the demand for three‐dimensional electron microscopy with high resolution. The ultrastructural change and related functional studies are necessary to investigate biological phenomena. In this study, currently available 3D reconstruction techniques of electron microscopes (serial block‐face scanning electron microscopy and focused ion beam—scanning electron microscopy) were used to investigate hyperpigmentary disorders in human skin. In the basal layer of the epidermis in the human skin, there are melanocytes that produce melanin and keratinocytes that act as a barrier against environmental damage. The 3D structure from serial images through scanning electron microscopy showed locations of melanosomes between melanocyte and keratinocyte in the hyperpigmentary disorder, in addition, the electron tomography showed pigment transfer through melanin instead melanosome. These results support the exocytosis‐endocytosis theory of pigment in human skin.     

Multiscale characterization of pathological bone tissue

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.     

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.     

Comparison of intensity distributions in tomograms from BF TEM,ADF STEM,HAADF STEM,and calculated tilt series

The three-dimensional (3D) morphology of a nanometer-sized object can be obtained using electron tomography. Variations in composition or density of the object cause variations in the reconstructed intensity. When imaging homogeneous objects, variations in reconstructed intensity are caused by the imaging technique, imaging conditions, and reconstruction. In this paper, we describe data acquisition, image processing, and 3D reconstruction to obtain and compare tomograms of magnetite crystals from bright field (BF) transmission electron microscopy (TEM), annular dark-field (ADF) scanning transmission electron microscopy (STEM), and high-angle annular dark field (HAADF) STEM tilt series. We use histograms, which plot the number of volume elements (voxels) at a given intensity vs. the intensity, to measure and quantitatively compare intensity distributions among different tomograms. In combination with numerical simulations, we determine the influence of maximum tilt angle, tilt increment, contrast changes with tilt (diffraction contrast), and the signal-to-noise ratio (SNR) as well as the choice of the reconstruction approach (weighted backprojection (WB) and sequential iterative reconstruction technique (SIRT)) on the histogram. We conclude that because ADF and HAADF STEM techniques are less affected by diffraction, and because they have a higher SNR than BF TEM, they are better suited for tomography of nanometer-sized crystals.     

Comparison of intensity distributions in tomograms from BF TEM, ADF STEM, HAADF STEM, and calculated tilt series

The three-dimensional (3D) morphology of a nanometer-sized object can be obtained using electron tomography. Variations in composition or density of the object cause variations in the reconstructed intensity. When imaging homogeneous objects, variations in reconstructed intensity are caused by the imaging technique, imaging conditions, and reconstruction. In this paper, we describe data acquisition, image processing, and 3D reconstruction to obtain and compare tomograms of magnetite crystals from bright field (BF) transmission electron microscopy (TEM), annular dark-field (ADF) scanning transmission electron microscopy (STEM), and high-angle annular dark field (HAADF) STEM tilt series. We use histograms, which plot the number of volume elements (voxels) at a given intensity vs. the intensity, to measure and quantitatively compare intensity distributions among different tomograms. In combination with numerical simulations, we determine the influence of maximum tilt angle, tilt increment, contrast changes with tilt (diffraction contrast), and the signal-to-noise ratio (SNR) as well as the choice of the reconstruction approach (weighted backprojection (WB) and sequential iterative reconstruction technique (SIRT)) on the histogram. We conclude that because ADF and HAADF STEM techniques are less affected by diffraction, and because they have a higher SNR than BF TEM, they are better suited for tomography of nanometer-sized crystals.     

Using the Hilbert transform for 3D visualization of differential interference contrast microscope images

Differential interference contrast (DIC) is frequently used in conventional 2D biological microscopy. Our recent investigations into producing a 3D DIC microscope (in both conventional and confocal modes) have uncovered a fundamental difficulty: namely that the phase gradient images of DIC microscopy cannot be visualized using standard digital image processing and reconstruction techniques, as commonly used elsewhere in microscopy. We discuss two approaches to the problem of preparing gradient images for 3D visualization: integration and the Hilbert transform. After applying the Hilbert transform, the dataset can then be visualized in 3D using standard techniques. We find that the Hilbert transform provides a rapid qualitative pre-processing technique for 3D visualization for a wide range of biological specimens in DIC microscopy, including chromosomes, which we use in this study.     

Surface and moisture characteristics of jute using a D.C. glow discharge argon plasma

A D.C. glow discharge plasma source was employed to characterize changes in water absorbance and surface morphology of natural jute fibers. The low temperature plasma removed moisture from the fibers and significantly modified surface properties. Scanning electron microscopy and energy dispersive X-ray spectroscopy were used for characterization. Open source software was used for the processing of the scanning electron micrographs. The changes in the macromolecular structure and the crystallinity were characterized by X-ray diffraction. Thermogravimetric analysis demonstrated moisture removal from the fibers following plasma treatment.     

蛋白质电子显微术

本文简要介绍了蛋白质电子显微术的基本原理和方法,包括低温电镜术和三维重构方法,以及该技术在研究膜蛋白二维晶体、二十面体对称性病毒和非对称性单颗粒蛋白质分子或复合物结构上的应用。随着分辨率的不断提高,蛋白质电子显微术已成为蛋白质结构测定的一种手段。     

Prospects for 3D, nanometer-resolution imaging by confocal STEM

Confocal STEM is a new electron microscopy imaging mode. In a microscope with spherical aberration-corrected electron optics, it can produce three-dimensional (3D) images by optical sectioning. We have adapted the linear imaging theory of light confocal microscopy to confocal STEM and use it to suggest optimum imaging conditions for a confocal STEM limited by fifth-order spherical aberration. We predict that current or near-future microscopes will be able to produce 3D images with 1 nm vertical resolution and sub-Angstrom lateral resolution. Multislice simulations show that we will need to be cautious in interpreting these images, however, as they can be complicated by dynamical electron scattering.     

A novel 3D wavelet-based filter for visualizing features in noisy biological data

We have developed a three-dimensional (3D) wavelet-based filter for visualizing structural features in volumetric data. The only variable parameter is a characteristic linear size of the feature of interest. The filtered output contains only those regions that are correlated with the characteristic size, thus de-noising the image. We demonstrate the use of the filter by applying it to 3D data from a variety of electron microscopy samples, including low-contrast vitreous ice cryogenic preparations, as well as 3D optical microscopy specimens.     

Automated high-throughput electron tomography by pre-calibration of image shifts

Electron tomography is a versatile method for obtaining three‐dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100–500 nm thick) with 4–20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several µm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five‐fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus‐change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.     

Comparison of manual and automatic processing of biological samples for electron microscopy

The ultrastructure of a sample can be observed by electron microscopy (EM), which has become an indispensable research tool in morphological studies. However, EM sample preparation techniques are complicated and time‐consuming, with a high labor cost. The current study was conducted to compare the conventional manual and automated methods for sample processing and post‐staining for electron microscopy. Automated sample processing reduces OsO₄ contamination, improves the efficiency of sample preparation and is easy to use. Therefore, the results of their study provide a practical and feasible method for the preparation of biological samples for electron microscopy.     

Retrieving modulation parameters from HRTEM images of modulated structures

Quantitative high-resolution transmission electron microscopy (qHRTEM) is methodologically extended towards the assessment of occupationally and positionally modulated structures. For this purpose, iterative digital image matching has been combined with data processing within the Rietveld refinement code JANA2000. In this approach, the number of free parameters is kept low and rather complicated modulated structures become assessable by qHRTEM. The feasibility of the improved methodology is demonstrated for the 1D modulated structure of Ba(2)TiGe(2)O(8).     

Optimal design and fabrication of three-dimensional calibration specimens for scanning probe microscopy

Micro-/nano-scale roughness specimens are highly demanded to synthetically calibrate the scanning probe microscopy (SPM) instrument. In this study, three-dimensional (3D) specimens with controllable main surface evaluation parameters were designed. In order to improve the design accuracy, the genetic algorithm was introduced into the conventional digital filter method. A primary 3D calibration specimen with the dimension of 10 μm × 10 μm was fabricated by electron beam lithography. Atomic force microscopy characterizations demonstrated that the statistical and spectral parameters of the fabricated specimen match well with the designed values. Such a kind of 3D specimens has the potential to calibrate the SPM for applications in quantitative surface evaluations.     

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells

A combination of two‐dimensional (2D) and three‐dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and β cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a β cell.     

Single molecule microscopy methods for the study of DNA origami structures

Single molecule microscopy techniques play an important role in the investigation of advanced DNA structures such as those created by the DNA origami method. Three single molecule microscopy techniques are particularly interesting for the investigation of complex self-assembled three-dimensional (3D) DNA nanostructures, namely single molecule fluorescence microscopy, atomic force microscopy (AFM), and cryogenic transmission electron microscopy (cryo-EM). Here we discuss the strengths of these three techniques and demonstrate how their interplay can yield very important and unique new insights into the structure and conformation of advanced biological nanostructures. The applications of the three single molecule microscopy techniques are illustrated by focusing on a self-assembled DNA origami 3D box nanostructure. Its size and structure were studied by AFM and cryo-EM, while the lid opening, which can be controlled by the addition of oligonucleotide keys, was recorded by F?rster/fluorescence resonance energy transfer (FRET) spectroscopy.     

Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

Stain density is an important parameter for optimising the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue and liver tissue.