Mounting Medium Research Articles

Pushing the limits of optical resolution in the study of the tiniest fossil arthropods

ABSTRACT Studies of minute fossils trapped in amber shed light on the chronology of species and allow us to track evolutionary, ecological and biospheric changes. However, the resolution of commonly used methods for imaging amber fossils is limited, and the quality of preservation is not the only reason for this limitation. We have used super-resolution confocal laser microscopy (Airyscan) to study fossil mites (Acari) from the Eocene and Cretaceous amber (35–100 Ma). Here we report our technical approaches and methodological findings, including the selection of mounting medium, clearing of specimens, the effects of hydration of fossils in amber and the advantages of super-resolution confocal microscopy in obtaining images with sub-micron detail. Our results show that modern microscopy, in combination with specific non-destructive methods of amber preparation, can facilitate achieving image resolutions that were previously hardly feasible in fossils.

Permanent Feulgen staining preparation without mounting medium use

Introduction. Feulgen staining is used in medical and biological studies when it is necessary to assess the level of DNA and its localization in cells. Some fixatives, mounting media and light can decrease the quality of preparations for microscopy by action on dye. Aim of the study. Our experiment aimed і) to develop the Feulgen procedure modification that excluded the use of harsh fixation agents and mounting media and іі) to evaluate visually the effect of periodical light exposure during teaching process within 6 years onto Feulgen stained preparations quality. Materials and methods. To escape the problem of stained specimens deterioration, we omitted the use of strong fixatives and mounting media, and avoided long light expositions on microscopic preparations. For permanent microscopic preparations production, the stained specimen covered with coverslip was properly air dried and then sealed by scotch tape on coverslip perimeter instead of mounting medium use. Preparations were kept wrapped in black paper and used for teaching of medical and biological students for eproximately 20 hours each year within a 6-years period. The microphotographs of the same site in tissue made at the start and end of this period were compared in quality visually. Results and discussion. Air-drying of stained specimens between the glass slide and coverslip without mounting medium followed by their sealing with scotch tape provided preparations of good quality. Their use for teaching of students within 6 years during eproximately 20 hours per year caused no fading or other visually detectable changes in stained tissue. This was proved from comparison of microphotographs made at the start and end of this period. Conclusion. Suggested variation of the Feulgen staining method can be applied for teaching of students in medicine and biology and, possibly, for routine analyses.

Supplementary files

Fig. S1. Phylogenetic tree resulting from ML analysis of the ITS gene region alignment. The confidence values are indicated at the nodes in the same way as in Fig. 7. Genus clades are discriminated with boxes in different colours and clades containing thermophilic species are highlighted with an orange background. The scale bar shows the expected number of changes per site. The tree is rooted with Pseudoechria longicollis and Schizotheciaceae, in the Schizotheciaceae.<br />Fig. S2. Phylogenetic tree resulting from ML analysis of the partial tub2 gene region alignment. The confidence values are indicated at the nodes in the same way as in Fig. 7. Genus clades are discriminated with boxes in different colours and clades containing thermophilic species are highlighted with an orange background. The scale bar shows the expected number of changes per site. The tree is rooted with Pseudoechria longicollis and Schizotheciaceae, in the Schizotheciaceae, Sordariales.<br />Fig. S3. Phylogenetic tree resulting from ML analysis of the partial rpb2 gene region alignment. The confidence values are indicated at the nodes in the same way as in Fig. 7. Genus clades are discriminated with boxes in different colours and clades containing thermophilic species are highlighted with an orange background. The scale bar shows the expected number of changes per site. The tree is rooted with Pseudoechria longicollis and Pseudoechria prolifica, in the Schizotheciaceae.<br />Fig. S4. Phylogenetic location of “Humicola koreana” based on the separate analyses of partial gene sequences of ITS (A) and LSU (B). The confidence values are indicated at the nodes in the same way as in Fig. 7. The scale bar shows the expected number of changes per site. The tree is rooted with Achaetomium strumarium.<br />Table S1. Comparison ascospore sizes measured in lactic acid (present study) and water (literature) as mounting medium.<br />Table S2. Overview species and strains used in phylogenetic analysis.

Click-iT ® Plus OPP Alexa Fluor ® Protein Synthesis Assay in Embryonic Cells.

This protocol describes a method to assess relative changes in the level of global protein synthesis in the preimplantation embryo using the Click-iT ® Plus OPP Protein Synthesis Assays. In this assay, O-propargyl-puromycin (OPP), an analog of puromycin, is efficiently incorporated into the nascent polypeptide of newly translated proteins in embryonic cells. OPP is fluorescently labeled with a photostable Alexa Fluor TM dye and detected with fluorescence microscopy. The intensity of the fluorescence is quantitatively analyzed. This is a fast, sensitive, and non-radioactive method for the detection of protein synthesis in early embryo development. It provides a tool for analyzing the temporal regulation of protein synthesis, as well as the effects of changes in the embryonic microenvironment, and pharmacological and genetic modulations of embryo development. Graphical abstract: Figure 1. Brief overview of the procedures of the Click-iT ® Plus OPP Alexa Fluor ® protein synthesis assay in embryonic cells. (A) Set up OPP treatments: (1) Set up microdrops containing 50 µL of OPP working solution and label different treatments on the back of culture dishes ( e.g. , T0, T1, T2, and T3); (2) The drops are overlain with 2-3 mm heavy paraffin oil and then equilibrated in incubator for 2 h; (3) Collect the embryos from female reproductive tracts or following in vitro culture in desired treatments; (4) Culture embryos in the equilibrated OPP working solution for 2-6 h. ( B ) Example of OPP detection procedures working with 60-well plates labeled as T0, T1, T2, T3, T4, and T5 for different treatments: (1) The first 60-well plate is used for the procedures of washing, fixation, permeabilization, and Click-iT ® OPP detection. (2) The second 60-well plate is for DNA staining and washing. (C) Slide preparation: (1) Label the required number of slides and set up vaseline coverslip supports; (2) Add mounting medium; (3) Transfer embryos into mounting medium; (4) Set coverslip; (5) Seal the coverslip with nail polish.

Tricks of the Trade | An Alternative to the Traditional Coverslip Gripper, Particle Disperser, and Crusher

When using glass slides, glass coverslips, and liquid mounting media for sample preparation in light microscopy, it is essential to have well-dispersed particles in order to facilitate the characterization and identification of individual particles. The traditional way to disperse or redistribute particles under the coverslip and flatten the mounting medium is by using the eraser end of an ordinary pencil as a means to “grip” the glass coverslip and move it around by hand pressure in a controlled manner using the pencil shaft as a handle. By pressing the eraser on the coverslip and sliding the coverslip in a circular pattern, particles are dispersed individually and the mounting medium is more easily flattened and spread throughout the underside of the coverslip.

Evaluation of New Immunohistochemical Approaches for the Study of Kidney Tumors in Geriatric.

Kidney malignancies are among the most deadly genitourinary tumors. It is more common in males and is often seen in people aged 60-70 years old. The incidence rate of kidney cancer seems to be increasing. One reason for this may be the fact that imaging techniques, such as computed tomography scans are more commonly used. These tests may lead to the accidental detection of more kidney cancers. Fortunately, kidney cancer is often detected in the early stages, when the tumor is small and confined to the kidney. The objective of this study was the development of new diagnostic immunohistochemical methods. Clinical examination material of 134 people, including 94 (70%) males and 40 (30%) females, were used in this study. Immunohistochemical staining of tryptase was carried out in compliance with the requirements using Anti-Mast Cell Tryptase antibodies. Goat anti-mouse antibodies #AS-M1-HRP were used as secondary antibodies, visualized with ImmPACTTM DAB Peroxidase Substrate Kit (#SK-4105) according to the instructions of the manufacturer. The nuclei were counterstained with Mayer's hematoxylin, and the sections were embedded in a permanent mounting medium.The immunohistochemical study showed an increase in both tryptase- and chymase-positive mast cells in the renal parenchyma, compared with the control group. The number of mast cells with tryptase expression directly in the tumor was significantly less than the peritumoral localization. A similar pattern was observed for chymase-positive mast cells as the content of the tumor was more than 10 times higher than the intratumoral arrangement. The histological and immunological characteristics did not differ in different age groups. The immunohistochemical method of research in the diagnosis of renal tumors plays an important diagnostic and prognostic value. It can assist pathologists in difficult and ambiguous cases to correctly diagnose renal tumors. This will make it possible to prescribe the correct treatment and predict the course of malignant tumor growth in patients.

Forensic hair analysis – Worldwide survey results

Forensic hair analysis remains under the microscope. Although hair evidence can yield information pertinent to an investigation, past issues with associated expert testimony endure. The aim of this survey was to gain a better understanding of hair analysts by expanding on past findings by assessing methodology, terminology and bias, analysing demographic and workplace data and allowing participants to comment on current and future issues in hair analysis. An online survey was designed and disseminated to hair analysts worldwide and 176 analysts from 12 countries took part in the survey. Participants responded to questions regarding accreditation, the probative value of hair evidence, collection and packaging, contextual information, methodology, terminology and reporting results, conclusions and opinions. The probative value of hair evidence was supported and other than selection of mounting medium the survey identified a preference of collection methods, packaging materials, use of microscopy and notetaking. Terminology used when conducting hair analysis and reporting results varied and caution towards statistics was expressed while training and collaboration was encouraged. The survey data will be used in the development of validation studies in hair analysis for future dissemination.

Megaspores from the Late Triassic‒Early Jurassic of southern Scandinavia: taxonomic and biostratigraphic implications

ABSTRACT Here we investigate megaspores from 10 Triassic‒Jurassic localities of southern Sweden and Bornholm, Denmark, based on collections housed in the Swedish Museum of Natural History. We identify and describe 19 megaspore taxa belonging to three stratigraphically constrained assemblages, representing the Rhaetian, Hettangian and Pliensbachian, respectively. Megaspores are abundant and diverse (12 taxa) in the Rhaetian assemblage. Diversity markedly decreases across the Triassic–Jurassic boundary (TJB), with species richness reducing from 12 to two taxa. The Hettangian assemblage is well-preserved but depauperate, and is overwhelmingly dominated by Nathorstisporites hopliticus. A subsequent recovery of lycopsid diversity followed, recorded by an increase in richness to six taxa in the Pliensbachian assemblage. The disappearance of the hygrophilous and diverse heterosporous lycophyte communities across the TJB, suggests a shift to drier conditions in the earliest Jurassic. This is supported by lithological changes from coal-forming environments in the Rhaetian to sandstone-dominated fluvial-estuarine facies in the Hettangian. Throughout this study, we analysed the megaspores using fluorescence microscopy, which revealed detailed morphological features on specimens that were otherwise opaque under visible light. This non-destructive technique is particularly useful for examining opaque megaspores embedded in permanent mounting media, such as epoxy resin, and may provide new insights into historical megaspore collections elsewhere.

Ageing Effects in Mounting Media of Microscope Slide Samples from Natural History Collections: A Case Study with Canada Balsam and PermountTM.

Microscope slide collections represent extremely valuable depositories of research material in a natural history, forensic, veterinary, and medical context. Unfortunately, most mounting media of these slides deteriorate over time, with the reason for this not yet understood at all. In this study, Raman spectroscopy, ultraviolet–visible (UV–Vis) spectroscopy, and different types of light microscopy were used to investigate the ageing behaviour of naturally aged slides from museum collections and the experimentally aged media of Canada balsam and Permount™, representing a natural and a synthetic resin, respectively, with both being based on mixtures of various terpenes. Whereas Canada balsam clearly revealed chemical ageing processes, visible as increasing colouration, Permount™ showed physical deterioration recognisable by the increasing number of cracks, which even often impacted a mounted specimen. Noticeable changes to the chemical and physical properties of these mounting media take decades in the case of Canada balsam but just a few years in the case of Permount™. Our results question whether or not Canada balsam should really be regarded as a mounting medium that lasts for centuries, if its increasing degree of polymerisation can lead to a mount which is no longer restorable.

Laser-free super-resolution microscopy.

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as 'blinking'), detected and localized. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Complex Photophysical Properties of K114 Make for a Versatile Fluorescent Probe for Amyloid Detection.

Protein aggregation is a hallmark of Alzheimer's disease (AD) and many other neurodegenerative disorders. Small organic fluorophores such as Congo Red preferentially bind to cross-β-sheet-rich deposits and have been used to label amyloid plaques and tau tangles in histological samples. However, distinguishing between different conformations of protein aggregates is not trivial. Using silkworm and spider silks (prototypical amyloids) and transgenic AD mouse (5XFAD) and human AD brain samples, we report how spectral confocal microscopy allowed for improved detection and differentiation of protein aggregates based on the unexpected photophysical behavior of the amyloid-specific dye K114. The pH and excitation power had pronounced effects on the emission spectrum and intensity of amyloid-bound K114 fluorescence. When bound to β-sheet-rich assemblies, the emission spectrum of K114 was governed by the local pH of the binding pockets much more than by the pH of the mounting medium, likely due to ionization of titratable phenols. Unexpectedly, exposure to high excitation power caused a permanent increase in fluorescence intensity and a spectral blue-shift. These light-induced fluorescence changes were dependent in a complex manner on laser power, exposure time, pH, and amyloid type examined. The above-mentioned phenomena were observed in silk fibers and Alzheimer brain sections from mouse and human, indicating that this may be a general characteristic of K114 when bound to tightly aggregated macromolecules. Potential mechanisms are discussed, likely involving photoinduced electron transfer. Our findings illustrate how the complex photophysical behavior of amyloid-bound K114 can be exploited for improved detection and differentiation of protein aggregates.

Variable immersion microscopy with a high numerical aperture.

Three-dimensional (3D) optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. We developed a variable immersion lens (VIL) to passively minimize these aberrations. A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror). Wave-optics simulation at a wavelength of 488 nm showed that a VIL microscope enables diffraction-limited 1.2-NA imaging in water (refractive index of 1.34) at a depth of 0.3 mm by minimizing aberrations due to refraction of a sample interface. Another aberration due to the refractive index mismatching between a mounting medium, and an object can also be corrected by the VIL system, because various fluids with different refractive indices can be used as mounting media for the VIL. As a result of correcting the two aberrations at the same time, we experimentally demonstrated that a 6 µm diameter fluorescent bead can be imaged to the true dimensions in 3D.

KOH Wet Mount versus Cellophane Strip without Mounting Medium for Rapid Diagnosis in Superficial Mycoses

Context: Diagnosis of superficial mycoses in clinically doubtful cases relies on demonstration of fungal elements on a 10% potassium hydroxide (KOH) slide mount which takes 30 min to 2 h. Rapid results will lead to reduced time to diagnosis. Aims: A comparative study of conventional 10% KOH wet mount against the cellophane strip method without a mounting medium under bright-field and phase-contrast microscopy was aimed to determine the comparative efficacy of these methods in demonstrating fungal elements of superficial mycoses. Settings and Design: The study was conducted at a skin center of a tertiary care hospital between November 2019 and January 2020. Material and Methods: One hundred and fifty consecutive clinically suspect cases of superficial cutaneous mycoses were examined. Skin scrapings for 10% KOH were collected with a blunt scalpel on one slide and examined half hour later. A cellophane strip collection of squames stuck onto another slide and examined directly by bright-field and phase-contrast microscopy. Statistical Analysis Used: Descriptive and inferential statistical analysis was carried out in the present study using SPSS 22.0, and R environment version 3.2.2 was used for the analysis of the data. Chi-square/Fisher's exact test was used to find the significance of study parameters on categorical scale between two or more groups, nonparametric setting for qualitative data analysis. Fisher's exact test was used when samples were very small. Results: The mean percentage positivity for fungal hyphae as seen on KOH mount was 76%, for cellophane tape 97.33%, and under phase-contrast microscopy 97.33%. Pityriasis versicolor was detected by all three methods (100%). Tinea corporis had the least positive result on KOH mount. Conclusion: Cellophane strip method without a mounting medium adequately demonstrates fungal elements on light microscopy.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

Practical Aspects of Super-Resolution Imaging and Segmentation of Macromolecular Complexes by dSTORM.

Super-resolution fluorescence microscopy allows imaging macromolecular complexes down to the nanoscopic scale and thus is a great tool to combine and integrate cellular imaging in the native cellular environment with structural analysis by X-ray crystallography or high-resolution cryoelectron microscopy or tomography. Here we describe practical aspects of SMLM imaging by dSTORM, from the initial sample preparation using mounting media, antibodies and fluorescent markers, the experimental setup for data acquisition including multi-color colocalization and 3D data acquisition, and finally tips and clues on advanced data processing that includes image reconstruction and data segmentation using 2D or 3D clustering methods. This approach opens the path toward multi-resolution integration in cellular structural biology.

Integrity of the blood‐brain barrier and changes in the microvasculature during the progression of Alzheimer’s disease in the 3XTG‐AD model

AbstractBackgroundThere is evidence that vasculature failures are related with the development of Alzheimer´s. Between 60 and 90% of patients with AD present with pathologies of cerebrovascular disease. The blood brain barrier (BBB) is a highly selective, semipermeable chemical and structural barrier that ensures a stable internal environment in the brain. BBB dysfunction was initially identified in animal models for AD, and confirmed in patients. To date it is not clear how it originates. Physically, BBB endothelial cells are bound by tight junction complexes (TJ), formed by plasma membrane proteins, such as ocludins and claudins and cytoplasmic scaffolding proteins, such as ZO1. The study of changes in the expression of TJ proteins in a 3xTg‐AD mouse model at different ages will allow us to determine if damage to the BBB occurs before the appearance of amyloid‐β plaques.MethodThe animal's brain (3xTg‐AD mice of 9 and 13 months) is removed. Subsequently, the microvasculature is precipitated by density‐dependent centrifugation through the use of a solution with dextran, managing to separate the cerebral parenchyma and the myelin from the microvasculature. Once the microvessels are obtained, we incubate with anti‐ZO‐1, claudin‐5 and ocludin fluorescent primary antibodies and secondary antibodies (Alexas 488 and 546) and Hoechst 33342. Isolated microvessels are mixed with a fluorescent mounting medium (Fluoromount), mounted on slides and covered with a coverslip. Once mounted, they are scanned with a confocal laser scanning microscope.ResultWe found a decrease in the expression of tight junction proteins in 3xTg‐AD mice of 13 months compared to the same model but at 9 months and also compared to control animals. Morphological differences were also observed in the microvessels of the animals 3xTg‐AD in comparison with the WT and between the animals of 9 and 13 months, with morphological damage being more evident in the older animals.ConclusionThis could be telling us that the BBB is compromised. We still have to analyze the association between this decrease in TJ proteins and the accumulation of beta‐amyloid, in addition to analyzing it at earlier ages in order to establish from what age these changes in this murine model are measurable.

Thrombin Generation Related to Netosis in Patients with Systemic Lupus Erythematosus

Thrombin Generation Related to Netosis in Patients with Systemic Lupus Erythematosus

Minimizing shrinkage of acute brain slices using metal spacers during histological embedding

The morphological structure of neurons provides the basis for their functions and is a major focus of contemporary neuroscience studies. Intracellular staining of single cells in acute slices is a well-established approach, offering high-resolution information on neuronal morphology, complementing their physiology. Despite major technical advances, however, a common histological artifact often precludes precise morphological analysis: shrinkage of the sampled tissue after embedding for microscopy. Here, we describe a new approach using a metal spacer, sandwiched between two coverslips to reduce shrinkage of whole-mount slice preparations during embedding with aqueous mounting medium under a coverslip. This approach additionally allows imaging the slices from both sides to obtain better quality images of deeper structures. We demonstrate that the use of this spacer system can efficiently and stably reduce the shrinkage of slices, whereas conventional embedding methods without spacer or with agar spacer cause severe, progressive shrinkage after embedding. We further show that the shrinkage of slices is not uniform and artifacts in morphology and anatomical parameters produced cannot be compensated using linear correction algorithms. Our study, thus, emphasizes the importance of preventing the deformation of slice preparations and offers an effective means for reducing shrinkage and associated artifacts during embedding.

A quantitative method for selecting a hair for nuclear DNA analysis

This study evaluated a quantitative method to predict the success of nuclear DNA (nuDNA) typing for head hair roots, using the minor-groove DNA binding dye, 4′, 6-diamidino-2-phenylindole (DAPI). The procedure was successful in staining nuclear material in hair roots, regardless of soft tissue presence or growth phase. We found that the dye can even reveal an abundance of visible nuclei in hairs that were previously assumed to be unsuitable for nuDNA analysis (e.g., telogen hairs). The value of DAPI staining is particularly evident when considering the STR typing results for telogen hairs. Here, telogen hairs with greater than 100 visible nuclei frequently produced full or high-partial STR profiles, while telogen hairs with fewer than 100 visible nuclei rarely resulted in >20 % STR allele recovery. In addition, our findings indicated no interference by DAPI in the forensic examination of hair evidence, including preparation of hairs on microscopic slides, microscopic examination, DNA extraction, quantitative PCR, and short tandem repeat (STR) typing. Furthermore, the method remained steadfast for hairs washed by sonication as well as hairs retrieved from Permount™ mounting medium. When validated, this simple, quick, and quantitative screening method can be used in casework to select a hair for nuDNA analysis, especially for hairs that were previously sent directly for mitochondrial (mt) DNA analysis based on the lack of adhering soft tissue, regardless of growth phase. Conversely, nuDNA degradation may exist in hairs which exhibit microscopic characteristics typically associated with a potential to generate successful nuclear DNA profile including stretched roots with attached root sheath. DAPI staining of hairs gives forensic examiners the ability to have more information, other than growth phase, when selecting a hair or hairs for possible nuDNA analysis.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.