A comparative study on the use of microscopy in pharmacology and cell biology research.

Microscopy is the main technique to visualize and study the structure and function of cells. The impact of optical and electron microscopy techniques is enormous in all fields of biomedical research. It is possible that different research areas rely on microscopy in diverse ways. Here, we analyzed comparatively the use of microscopy in pharmacology and cell biology, among other biomedical sciences fields. We collected data from articles published in several major journals in these fields. We analyzed the frequency of use of different optical and electron microscopy techniques: bright field, phase contrast, differential interference contrast, polarization, conventional fluorescence, confocal, live cell imaging, super resolution, transmission and scanning electron microscopy, and cryoelectron microscopy. Our analysis showed that the use of microscopy has a distinctive pattern in each research area, and that nearly half of the articles from pharmacology journals did not use any microscopy method, compared to the use of microscopy in almost all the articles from cell biology journals. The most frequent microscopy methods in all the journals in all areas were bright field and fluorescence (conventional and confocal). Again, the pattern of use was different: while the most used microscopy methods in pharmacology were bright field and conventional fluorescence, in cell biology the most used methods were conventional and confocal fluorescence, and live cell imaging. We observed that the combination of different microscopy techniques was more frequent in cell biology, with up to 6 methods in the same article. To correlate the use of microscopy with the research theme of each article, we analyzed the proportion of microscopy figures with the use of cell culture. We analyzed comparatively the vocabulary of each biomedical sciences field, by the identification of the most frequent words in the articles. The collection of data described here shows a vast difference in the use of microscopy among different fields of biomedical sciences. The data presented here could be valuable in other scientific and educational contexts.

Electron microscopy study of 496 cases of lupus nephritis: A single-center experience.

INTRODUCTION: Renal involvement is seen in about 40-82% of systemic lupus erythematosus (SLE) Asian patients. The exact diagnosis and classification of lupus nephritis are important for treatment and prognosis. This study aimed to investigate the value of electron microscopy (EM) in the diagnosis and classification of lupus nephritis compared with light microscopy. METHOD: In this cross-sectional referral-center 16-year study of lupus nephritis, the final diagnosis was based on the EM study. Primary light microscopy findings were compared with EM diagnosis. Moreover, Immunofluorescence patterns distribution was assessed. RESULTS: From 496 patients diagnosed with lupus nephritis based on EM, 225(45.4%) of patients were categorized in class IV, followed by 98(19.7%), 93(18.8%), 46(9.3%), and 14(2.8%) who were categorized into classes of II, III, V, and VI respectively. Only 1(0.2%) patient belonged to class I, and 19(3.8%) cases were diagnosed with mixed two classes. Using EM was essential for diagnosing 25.6% of cases taking the correct classification by light microscopy into account; however, disregarding correct classification, this could change to a 7.4% contribution rate of EM. The most common cause of misdiagnosis, disregarding incorrect classification, was inadequate or wrong tissue. Positive associations were detected between tubular atrophy and interstitial fibrosis of both electron and light microscopy with different classes (P < 0.001). CONCLUSION: While light microscopy is highly accurate for diagnosing lupus nephritis regardless of correct classification, EM contributes substantially to the correct classification of lupus nephritis types.

A 16-year Survey of Clinicopathological Findings, Electron Microscopy, and Classification of Renal Amyloidosis.

Background: Electron microscopy (EM) is a valuable tool in the diagnosis of renal amyloidosis, particularly in the early stages of the disease. In Iran, studies on EM and the clinical features of renal amyloidosis are scarce. The objective of the present study was to survey EM investigations, pathological classifications, and clinical features of renal amyloidosis. Methods: This cross-sectional study was performed in Shiraz, Iran, during 2001-2016. Out of 2,770 kidney biopsies, 27 cases with a diagnosis of renal amyloidosis were investigated. EM investigation and six staining procedures for light microscopy (LM) were performed. Two pathological classifications based on glomerular, peritubular, perivascular, and interstitial involvement were made. Finally, the association between these classifications and the clinical features was assessed. Chi-square, Fisher's exact, Independent t test, and Multiple logistic regression analysis were used. P values<0.05 were considered statistically significant. Results: In 51.9% of the cases, the clinical diagnosis was nephrotic syndrome. Proteinuria and edema were the most prevalent clinical manifestations. The role of EM investigation for diagnosis was graded "necessary" or "supportive" in 48.2% of the patients. In the classification based on glomerular classes, variables such as the mean blood pressure (P=0.003), history of hypertension (P=0.02), creatinine >1.5 (P=0.03), and severe tubular atrophy (P=0.03) were significantly higher in class B (advanced amyloid depositions). Conclusion: EM plays an important role in the diagnosis of renal amyloidosis. EM in conjunction with LM investigation with Congo red staining is recommended, to prevent misdiagnosis of patients with a clinical suspicion of renal amyloidosis. Among different pathological features of renal amyloidosis, the severity of glomerular amyloid depositions had a clear relationship with clinical presentations.

A nationwide survey on clinical practice patterns and bleeding complications of percutaneous native kidney biopsy in Japan.

BACKGROUND: Practice patterns and bleeding complications of percutaneous native kidney biopsy (PNKB) have not recently been investigated and the Japanese Society of Nephrology performed a nationwide questionnaire survey in 2018. METHODS: The survey consisted of nine sections about PNKB: (1) general indications; (2) indications for high-risk patients; (3) informed consent; (4) pre-biopsy evaluation; (5) procedures; (6) sedation; (7) post-biopsy hemostasis, bed rest, and examinations; (8) bleeding complications; and (9) specimen processing. A supplementary survey examined bleeding requiring transcatheter arterial embolization (TAE). RESULTS: Overall, 220 directors of facilities (nephrology facility [NF], 168; pediatric nephrology facility [PF], 52) completed the survey. Indications, procedures, and monitoring protocols varied across facilities. Median lengths of hospital stay were 5 days in NFs and 6 days in PFs. Gauge 14, 16, 18 needles were used in 5%, 56%, 33% in NFs and 0%, 63%, 64% in PFs. Mean limits of needle passes were 5 in NFs and 4 in PFs. The bed rest period was 16-24 h in 60% of NFs and 65% of PFs. Based on 17,342 PNKBs, incidence rates of macroscopic hematuria, erythrocyte transfusion, and TAE were 3.1% (NF, 2.8%; PF, 6.2%), 0.7% (NF, 0.8%; PF, 0%), and 0.2% (NF, 0.2%; PF, 0.06%), respectively. Forty-six percent of facilities processed specimens all for light microscopy, immunofluorescence, and electron microscopy, and 21% processed for light microscopy only. Timing of bleeding requiring TAE varied among PNKB cases. CONCLUSION: Wide variations in practice patterns of PNKB existed among facilities, while PNKBs were performed as safely as previously reported.

Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.

While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available.

Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdb.org), which includes the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Data Bank Japan (PDBj; pdbj.org), the Protein Data Bank in Europe (PDBe; pdbe.org), and BioMagResBank (BMRB; www.bmrb.wisc.edu). The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (deposit.wwpdb.org). The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates. wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources. At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc.) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods.

TED: A Tolerant Edit Distance for segmentation evaluation.

In this paper, we present a novel error measure to compare a computer-generated segmentation of images or volumes against ground truth. This measure, which we call Tolerant Edit Distance (TED), is motivated by two observations that we usually encounter in biomedical image processing: (1) Some errors, like small boundary shifts, are tolerable in practice. Which errors are tolerable is application dependent and should be explicitly expressible in the measure. (2) Non-tolerable errors have to be corrected manually. The effort needed to do so should be reflected by the error measure. Our measure is the minimal weighted sum of split and merge operations to apply to one segmentation such that it resembles another segmentation within specified tolerance bounds. This is in contrast to other commonly used measures like Rand index or variation of information, which integrate small, but tolerable, differences. Additionally, the TED provides intuitive numbers and allows the localization and classification of errors in images or volumes. We demonstrate the applicability of the TED on 3D segmentations of neurons in electron microscopy images where topological correctness is arguable more important than exact boundary locations. Furthermore, we show that the TED is not just limited to evaluation tasks. We use it as the loss function in a max-margin learning framework to find parameters of an automatic neuron segmentation algorithm. We show that training to minimize the TED, i.e., to minimize crucial errors, leads to higher segmentation accuracy compared to other learning methods.

Prognostic Value of a Category Based on Electron Microscopic Features of Clival Chordomas.

OBJECTIVE: Skull base chordomas are clinically malignant because of the difficulty of total removal and the high recurrence rate. Because the disease-free survival after surgery is currently unpredictable, there is a need for new parameters, obtained from histologic analyses of the resection specimen, that allows a risk stratification of patients with chordoma. METHODS: In recent years, electron microscopic diagnoses were introduced into the clinical practice for the diagnosis of chordoma in our department. Clinical outcomes and electron microscopic features were retrospectively reviewed in the study. The electron micrograph shows that clival chordoma can be divided into cell-dense type (CDT) and matrix-rich type (MRT). Of all the patients with chordoma, complete data from 27 patients were obtained. There were 12 patients in the CDT group and 15 patients in the MRT group. The paraffin-embedded tissue samples were stained with Ki-67 antibody. The prognostic values of electron microscopic classification were compared between the 2 groups. RESULTS: There were no statistical differences in the gender (P = 0.704) and age distribution (P = 0.243) between the 2 groups. There was also no statistical difference concerning the constitution of primitive tumors and recurrent tumors between the 2 groups (P = 0.706). The CDT group had a higher mortality rate than the MRT group (P = 0.037). The tumors in the CDT group were prone to recurrence and the need for reoperation within 1 year after surgery, which is statistically different from that in the MRT group (P < 0.001). Chordoma tumors of 23 patients (85.2%) stained positive for Ki-67. CDT chordomas had a higher Ki-67 proliferation index than the MRT chordomas (P = 0.013). CONCLUSIONS: The present study demonstrates the utility of the ultrastructural features in the prognostic outcome of patients with chordoma. According to the ultrastructures of chordomas, they can be divided into CDT and MRT. CDT chordoma cells have a more aggressive proliferative ability. Patients with CDT have a poor prognostic factor in clival chordoma, which has a higher risk of recurrence and a shorter survival.

Macromolecular confinement of therapeutic protein in polymeric particles for controlled release: insulin as a case study

ABSTRACT Sustained release systems for therapeutic proteins have been widely studied targeting to improve the action of these drugs. Molecular entrapping of proteins is particularly challenging due to their conformational instability. We have developed a micro-structured poly-epsilon-caprolactone (PCL) particle system loaded with human insulin using a simple double-emulsion w/o/w method followed by solvent evaporation method. This formulation is comprised by spheric-shaped microparticles with average size of 10 micrometers. In vitro release showed a biphasic behavior such as a rapid release with about 50% of drug delivered within 2 hours and a sustained phase for up to 48 h. The subcutaneous administration of microencapsulated insulin showed a biphasic effect on glycemia in streptozotocin-induced diabetic mice, compatible with short and intermediate-acting behaviors, with first transition peak at about 2 h and the second phase exerting effect for up to 48h after s.c. administration. This study reveals that a simplified double-emulsion system results in biocompatible human-insulin-loaded PCL microparticles that might be used for further development of optimized sustained release formulations of insulin to be used in the restoration of hormonal levels.

A novel low energy electron microscope for DNA sequencing and surface analysis.

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts. The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence.

Atomic structure from large-area, low-dose exposures of materials: a new route to circumvent radiation damage.

Beam-induced structural modifications are a major nuisance in the study of materials by high-resolution electron microscopy. Here, we introduce a new approach to circumvent the radiation damage problem by a statistical treatment of large, noisy, low-dose data sets of non-periodic configurations (e.g. defects) in the material. We distribute the dose over a mixture of different defect structures at random positions and with random orientations, and recover representative model images via a maximum likelihood search. We demonstrate reconstructions from simulated images at such low doses that the location of individual entities is not possible. The approach may open a route to study currently inaccessible beam-sensitive configurations.

Current and future delivery of diagnostic electron microscopy in the UK: results of a national survey.

AIMS: Electron microscopy (EM) remains essential to delivering several specialist areas of diagnosis, especially the interpretation of native renal biopsies. However, there is anecdotal evidence of EM units struggling to survive, for a variety of reasons. The authors sought to obtain objective evidence of the extent and the causes of this problem. METHODS: An online survey was undertaken of Fellows of the Royal College of Pathologists who use EM in diagnosis. RESULTS: A significant number of EM units anticipate having to close and hence outsource their EM work in the coming years. Yet most existing units are working to full capacity and would be unable to take on the substantial amounts of extra work implied by other units outsourcing their needs. Equipment and staffing are identified by most EM units as the major barriers to growth and are also the main reasons cited for units facing potential closure. CONCLUSIONS: In the current financial climate it seems unlikely that units will be willing to make the large investment in equipment and staff needed to take on extra work, unless they can be reasonably confident of an acceptable financial return as a result of increased external referral rates. The case is thus made for a degree of national coordination of the future provision of this specialist service, possibly through the National Commissioning Group or the new National Commissioning Board. Without this, the future of diagnostic EM services in the UK is uncertain. Its failure would pose a threat to good patient care.

Histologic and morphologic effects of valproic acid and oxcarbazepine on rat uterine and ovarian cells.

PURPOSE: To determine the histologic and morphologic effects of valproic acid (VPA) and oxcarbazepine (OXC) on rat uterine and ovarian cells. METHODS: Fifty-six female prepubertal Wistar rats (21-24 days old and weighing between 47.5 and 58.1 g) were divided equally into four groups, which were given drinking water (controls), 300 mg/kg/day of VPA, 100 mg/kg/day of OXC or VPA + OXC via gavage, for 90 days. Ovaries and uteri of rats on proestrous and diestrous phases of estrous cycle were extirpated and placed in a fixation solution. The tissue specimens were assessed with apoptosis (TUNEL) staining protocols, eosinophil counting, and electron microscopic techniques. RESULTS: In uteri, apoptosis in stroma, mitochondrial swelling, and cristolysis were observed in the VPA group, and OXC led to negative effects on epithelial cell and intracellular edema. In ovaries, both drugs increased apoptosis and intracytoplasmic edema. Organelle structure disruption was also observed in the OXC group. More conspicuous degenerative modifications were determined in the VPA + OXC group. In uteri, the number of TUNEL-positive luminal epithelial cells was 7.20 +/- 1.32 in controls, and significantly increased to 29.60 +/- 1.58, 34.20 +/- 2.53, and 54.80 +/- 2.04 in VPA, OXC, and VPA + OXC groups, respectively (p < 0.001). The highest number of TUNEL-positive glandular epithelium cells was observed in the VPA + OXC group; however, the number of TUNEL-positive stroma cells was highest in the VPA group. The highest number of eosinophils in stroma was in the VPA group. CONCLUSION: VPA and OXC trigger apoptotic and degenerative effects on rat uterine and ovarian cells. VPA also prevents implantation of embryo to the uterus and causes abortion via endometrial eosinophil infiltration.

Image processing for electron microscopy single-particle analysis using XMIPP.

We describe a collection of standardized image processing protocols for electron microscopy single-particle analysis using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through maximum-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with minimum efforts in learning about the details of this comprehensive package. Examples of applications are presented for a negative stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.

Classification of heterogeneous electron microscopic projections into homogeneous subsets.

The co-existence of different states of a macromolecular complex in samples used by three-dimensional electron microscopy (3D-EM) constitutes a serious challenge. The single particle method applied directly to such heterogeneous sets is unable to provide useful information about the encountered conformational diversity and produces reconstructions with severely reduced resolution. One approach to solving this problem is to partition heterogeneous projection set into homogeneous components and apply existing reconstruction techniques to each of them. Due to the nature of the projection images and the high noise level present in them, this classification task is difficult. A method is presented to achieve the desired classification by using a novel image similarity measure and solving the corresponding optimization problem. Unlike the majority of competing approaches, the presented method employs unsupervised classification (it does not require any prior knowledge about the objects being classified) and does not involve a 3D reconstruction procedure. We demonstrate a fast implementation of this method, capable of classifying projection sets that originate from 3D-EM. The method's performance is evaluated on synthetically generated data sets produced by projecting 3D objects that resemble biological structures.

Single-particle reconstruction from EM images of helical filaments.

Helical filaments were the first structures to be reconstructed in three dimensions from electron microscopic images, and continue to be extensively studied due to the large number of such helical polymers found in biology. In principle, a single image of a helical polymer provides all of the different projections needed to reconstruct the three-dimensional structure. Unfortunately, many helical filaments have been refractory to the application of traditional (Fourier-Bessel) methods due to variability, heterogeneity, and weak scattering. Over the past several years, many of these problems have been surmounted using single-particle type approaches that can do substantially better than Fourier-Bessel approaches. Applications of these new methods to viruses, actin filaments, pili and many other polymers show the great advantages of the new methods.

Use of Autostitch for automatic stitching of microscope images.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.

Automatic TEM image alignment by trifocal geometry.

Here we propose a novel method for automatic, markerless, feature-based alignment of TEM images suitable for electron tomography. The proposed method, termed trifocal alignment, is more accurate than the previous markerless methods. The key components developed are: (1) a reliable multi-resolution algorithm for matching feature points between images; (2) a robust, maximum-likelihood-based estimator for determining the geometry of three views--the trifocal constraint--required for validating the correctness of the matches; and (3) a robust, large-scale optimization framework to compute the alignment parameters from hundreds of thousands of feature point measurements from a few hundred images. The ability to utilize such a large number of measurements successfully compensates for point localization errors. The method was experimentally confirmed with electron tomography tilt series of biological and material sciences samples, consisting of from 40 to 150 images. The results show that, with this feature-based alignment approach, a level of accuracy comparable with fiducial marker alignment can be achieved.

Tilt-series and electron microscope alignment for the correction of the non-perpendicularity of beam and tilt-axis.

In electron tomography the sample is tilted in the electron microscope and projections are recorded at different viewing angles. In the correct geometric setting, the tilt-axis of the object under scrutiny is perpendicular to the beam direction. However, we will demonstrate that this does not necessarily apply to all electron microscopes equipped with the default column alignment. The resulting effect is that a conical tilt is performed, which has to be considered in the reconstruction to avoid artifacts and to improve the resolution. A novel solution, with significantly improved convergence properties, will be introduced for calculating the three-dimensional marker model, which is necessary for the alignment of the tilt-series. Thereby, the angle between the beam direction and the tilt-axis is calculated, together with other geometrical distortions, like magnification and rotation changes, and incorporated in the reconstruction. Hereby, artifacts can be eliminated at the image processing basis, and the resolution can be significantly improved at the medium to high range frequencies. Synthetical and real data are used to demonstrate the obstructions caused by this effect and the quality improvement of the reconstructions. Finally, we also present a way to align the hardware of the microscope to correct for the non-perpendicularity between the beam direction and the tilt-axis, which is specifically tailored for tomographic applications.