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Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.

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Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

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A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular membrane compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cells. Herein, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.

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Light microscopy has been a favorite tool of biological studies for almost a century, recently producing detailed images with exquisite molecular specificity achieving spatial resolution at nanoscale. However, light microscopy is insufficient to provide chemical information as a standalone technique. An increasing amount of evidence demonstrates that optical photothermal infrared microspectroscopy (O-PTIR) is a valuable imaging tool that can extract chemical information to locate molecular structures at submicron resolution. To further investigate the applicability of sub-micron infrared microspectroscopy for biomedical applications, we analyzed the contribution of substrate chemistry to the infrared spectra acquired from individual neurons grown on various imaging substrates. To provide an example of correlative immunofluorescence/O-PTIR imaging, we used immunofluorescence to locate specific organelles for O-PTIR measurement, thus capturing molecular structures at the sub-cellular level directly in cells, which is not possible using traditional infrared microspectroscopy or immunofluorescence microscopy alone.

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Advances in cellular reprogramming have radically increased the use of patient-derived cells for neurological research in vitro. However, adherence of human neurons on tissue cultureware is unreliable over the extended periods required for electrophysiological maturation. Adherence issues are particularly prominent for transferable glass coverslips, hindering imaging and electrophysiological assays. Here, we assessed thin-film plasma polymer treatments, polymeric factors, and extracellular matrix coatings for extending the adherence of human neuronal cultures on glass. We find that positive-charged, amine-based plasma polymers improve the adherence of a range of human brain cells. Diaminopropane (DAP) treatment with laminin-based coating optimally supports long-term maturation of fundamental ion channel properties and synaptic activity of human neurons. As proof of concept, we demonstrated that DAP-treated glass is ideal for live imaging, patch-clamping, and optogenetics. A DAP-treated glass surface reduces the technical variability of human neuronal models and enhances electrophysiological maturation, allowing more reliable discoveries of treatments for neurological and psychiatric disorders.

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Asbestos fibre counting by phase-contrast microscope is subject to many sources of variation, including those dependent on the analyst. In this study, asbestos sample slides prepared with relocatable coverslips have been used for fibre counting among voluntary analysts to evaluate their proficiency. One slide of amosite and one of chrysotile were distributed to all the analysts, and three proficiency testing rounds were conducted for amosite and four for chrysotile. Each relocatable coverslip has a report in which are reported for each viewing field both the number of certified fibres (Verified Fibres) and a drawing representing the shape and position of the individual fibres. In the first round, the analysts were asked to report only the number of fibres counted in each of the predesignated fields of view. In the other rounds, subsequently developed, the analysts had to report the number and the position of the fibres for each field. The reported number of fibres and their position in each of the designed fields were evaluated against their respective verified fibres, to identify types of error. Discrepancies between reported fibres and verified fibres in each field of view have been used to evaluate the proficiency of the analysts. The discrepancies can be positive (D+) or negative (D-) depending on whether the analyst counts, for a specific field of view, more or less fibres compared to the verified fibres. The score is calculated using the following equation: Score = (1 - ∑D+ + ∑│D-│/VF) × 100. An analyst obtaining a score of ≥60, which corresponds to (∑D+ + ∑│D-│)/VF ≤ 0.40, is proficient. The number of laboratories that participating in this study varied from 13 to 17 depending on the rounds. For amosite fibre counts, the results were generally good compared to a proficiency score of 60. The major error made by analysts was the counting of fibres shorter than 5 µm, where this error was of 62% of extra fibres and accounted for 8% over-estimation of amosite fibres. For chrysotile, a score of ≥50 has been used to consider an analyst as proficient. The results of chrysotile fibres showed that in the first round all analysts counted less than fifty per cent of the verified fibres. In the second round 10 analysts out of 13 reached a score of ≥50, 8 of 16 in the third and 10 of 12 in the fourth. For chrysotile fibres, the error relating to the counting of fibres shorter than 5 µm was of 56% of extra fibres, but the error that most influenced the results was the number of oversight-missing fibres. This type of error accounted for 97% of the missing fibres and for the 29% under-estimation of the chrysotile fibres. For amosite fibre counting, results of this study show an improvement of the analyst's performance. For the chrysotile fibre count, although there is a significant improvement in the comparison between some rounds, this is not continuous over time.

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Round, small-sized coverslips were coated for the first time with thin layers of indium tin oxide (ITO, 10-40 nm)/gold (Au, 2-8 nm) and annealed at 550 °C for several hours. The resulting nanostructures on miniaturized substrates were further optimized for the localized surface plasmon resonance (LSPR) chemosensing of a model molecule-1,2-bis-(4-ppyridyl)-ethene (BPE)-with a detection limit of 10-12 M BPE in an aqueous solution. All the fabrication steps of plasmonic-annealed platforms were characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM).

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The clinical translation of Fourier transform infrared (FT-IR) microspectroscopy in pathology will require bringing this technique as close as possible to standard practice in pathology departments. An important step is sample preparation for both FT-IR microspectroscopy and pathology. This should entail minimal disruption of standard clinical practice while achieving good quality FT-IR spectral data. In fact, the recently described possibility of obtaining FT-IR spectra of cells placed on glass substrates brings FT-IR microspectroscopy closer to a clinical application. We have now furthered this work in order to identify two different types of lung cancer cells placed on glass coverslips. Two types of sample preparation which are widely used in pathology, cytospin and smear, have been used. Samples were fixed with either methanol, used in pathology, or formalin (4% paraformaldehyde) used widely in spectroscopy. Fixation with methanol (alcohol-based fixative) removed lipids from cells causing a decrease in intensity of the peaks at 2850 cm-1 and 2920 cm-1. Nevertheless, we show for the first time that using either type of sample preparation and fixation on thin glass coverslips allowed to differentiate between two different types of lung cancer cells using either the lipid region or the fingerprint region ranging from 1800 cm-1 to 1350 cm-1. We believe that formalin-fixed cytospin samples would be preferred to study cells on thin coverslips using FT-IR microspectroscopy. This work presents a clear indication for future advances in clinical assessment of samples within pathology units to gain a deeper understanding of cells/tissues under investigation.

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The rising incidence of cancer worldwide is causing an increase in the workload in pathology departments. This, coupled with advanced analysis methodologies, supports a developing need for techniques that could identify the presence of cancer cells in cytology and tissue samples in an objective, fast, and automated way. Fourier transform infrared (FT-IR) microspectroscopy can identify cancer cells in such samples objectively. Thus, it has the potential to become another tool to help pathologists in their daily work. However, one of the main drawbacks is the use of glass substrates by pathologists. Glass absorbs IR radiation, removing important mid-IR spectral data in the fingerprint region (1800 cm-1 to 900 cm-1). In this work, we hypothesized that, using glass coverslips of differing compositions, some regions within the fingerprint area could still be analyzed. We studied three different types of cells (peripheral blood mononuclear cells, a leukemia cell line, and a lung cancer cell line) and lymph node tissue placed on four different types of glass coverslips. The data presented here show that depending of the type of glass substrate used, information within the fingerprint region down to 1350 cm-1 can be obtained. Furthermore, using principal component analysis, separation between the different cell lines was possible using both the lipid region and the fingerprint region between 1800 cm-1 and 1350 cm-1. This work represents a further step towards the application of FT-IR microspectroscopy in histopathology departments.

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In this study, stable gold nanoparticles (AuNPs) are fabricated for the first time on commercial ultrafine glass coverslips coated with gold thin layers (2 nm, 4 nm, 6 nm, and 8 nm) at 25 °C and annealed at high temperatures (350 °C, 450 °C, and 550 °C) on a hot plate for different periods of time. Such gold nanostructured coverslips were systematically tested via surface enhanced Raman spectroscopy (SERS) to identify their spectral performances in the presence of different concentrations of a model molecule, namely 1,2-bis-(4-pyridyl)-ethene (BPE). By using these SERS platforms, it is possible to detect BPE traces (10-12 M) in aqueous solutions in 120 s. The stability of SERS spectra over five weeks of thiol-DNA probe (2 µL) deposited on gold nano-structured coverslip is also reported.

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BACKGROUND: Microscope chambers that accept glass coverslips with cultured cells are often used to monitor intracellular Ca2+ concentration ([Ca2+]i) during cell superfusion. Unfortunately, the experimental maneuvers associated with the coverslip installation in these chambers (medium removal and re-application) trigger unintended [Ca2+]i elevations. NEW METHOD: To prevent these [Ca2+]i elevations, a Petri dish insert has been constructed. The insert features a superfusion-optimized well to grow cell cultures. After this insert is removed from the Petri dish, the well retains the medium. This feature allows the inserts to be installed in microscope chambers while keeping the cells submerged at all times. RESULTS: These inserts were used to test the impact of a transient medium removal from the well (an equivalent of a coverslip removal from the medium) on [Ca2+]i in primary murine cortical neurons and astrocytes, and in HEK-293 cells. In all of these models, the medium removal/re-application caused a micromolar [Ca2+]i spike. While in neurons this spike was caused by a Ca2+ influx, in astrocytes and HEK-293 cells, it was caused by a Ca2+ release from intracellular stores. After the spike, a subpopulation of neurons failed to restore low [Ca2+]i; in 24% of the astrocytes, the spike triggered [Ca2+]i oscillations. However, prior to the spike, [Ca2+]i was low and uniform in all these cells. COMPARISON WITH EXISTING METHOD(S): The new method avoids the artificially-induced [Ca2+]i elevations that take place during the handling of glass coverslips with cultured cells. CONCLUSIONS: The new method allows monitoring [Ca2+]i without disturbing the basal [Ca2+]i levels.

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The aim of this study was to evaluate the effect of parenteral administration of dexamethasone and levamisole on the accumulation of macrophages and formation of giant cells in chronic inflammation by foreign body and blood parameters in pacu (Piaractus mesopotamicus). It was used 50 mg/kg of levamisole and 2.0 mg/kg of dexamethasone and the combination of both drugs. Coverslips were implanted under the skin. After 2, 7, and 15 days postimplantation (DPI), fish were anesthetized for removal of coverslips and the number of macrophages and giant cells were count. It was also collected blood from the caudal vessel to evaluate: red blood cell count, hematocrit, hemoglobin concentration, leukocytes count, thrombocytes count, MCV, and MCHC. We observed that dexamethasone affects negatively the formation of giant cells in the chronic inflammation for foreign body. Levamisole, despite being immunostimulatory in several species, showed limited action. However, it was enough to counteract the effect of dexamethasone; the association of the drugs did not interfere significantly in erythrocyte and leukocyte number in most of the treatments and times studied. In dexamethasone group there was a reduction in the number of erythrocytes and hemoglobin associated with increased mean corpuscular volume, suggesting slight macrocytic anemia. At 15 DPI, most groups showed the recovery of hematologic response. As in mammals, dexamethasone affects negatively the inflammatory response. Levamisole showed little effect by itself. However, in some parameters the association of both drugs causes similar response to control and naïve groups, showing the antagonistic effect of these drugs.

O objetivo deste estudo foi avaliar o efeito da administração parenteral de dexametasona e levamisol na acumulação de macrófagos e formação de células gigantes na inflamação crônica por corpo estranho e avaliação de parâmetros sanguíneos em pacu (Piaractus mesopotamicus). Utilizou-se 50 mg/kg de levamisol e 2,0 mg / kg de dexametasona e a combinação de ambos fármacos. As lamínulas foram implantadas sob a pele. Depois de 2, 7 e 15 dias pós-implantação (DPI), os peixes foram anestesiados para a remoção das lamínulas e contagem do número de macrófagos e células gigantes. Foi coletado sangue da veia caudal para realizar contagem de células vermelhas, leucócitos, trombócitos, concentração de hemoglobina, MCV e MCHC. Observou-se que a dexametasona afeta negativamente a formação de células gigantes na inflamação crónica por corpo estranho. O levamisol, apesar de ser considerado imunoestimulante em várias espécies, mostrou ação limitada. No entanto, foi suficiente para neutralizar o efeito da dexametasona; a associação das drogas não interferiu significativamente no número de eritrócitos e de leucócitos na maioria dos tratamentos e períodos estudados. No grupo da dexametasona, houve redução do número de eritrócitos e concentração de hemoglobina associado ao aumento do volume corpuscular médio sugerindo leve anemia macrocítica. Aos 15 DPI, a maioria dos grupos mostrou recuperação na resposta hematológica. Como em mamíferos, a dexametasona afeta negativamente a resposta inflamatória. O levamisol mostrou pouco efeito por si só. No entanto, em alguns parâmetros a associação das duas drogas provoca resposta similar ao grupo controle e naive, mostrando o efeito antagonista de estas drogas.

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Microscopy is a simple, direct technique for examining the morphology of cells and their organelles. Cells are immobilized on a solid support that is optically suitable for microscopy, and then fixed. When coupled with antibody-based immunofluorescent or immunocytochemical methods, the expression of specific proteins can be quantified and localized. Specialized stains and dyes can also be used to visualize nucleic acids or other cellular structures. See alternative protocols for fixation of suspension cells on Preparation of Cells for Microscopy using Cytospin and Preparation of Cells for Microscopy using 'Cell Blocks'.

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In high throughput microscopy, it is often assumed that the objects under investigation are fixed spatially. In addition, it is also presumed that the objects are sufficiently populated, otherwise there will be need to search through vast tracks of field of views before any recording can be done. The ability to collect objects at one location in the hydrated state is thus desirable and this is a challenge when the density of target objects in a sample is very low. In this work, we report that the generation of a squeezing flow from a circular coverslip compressing on suspensions is able to collect particulate (microbeads, fluorescent nanobeads and live algal cells) and non-particulate (EGFP) objects at the rim region of the coverslip. With a coverslip of 13 mm diameter, volumes between 2 µL and 4 µL were found to completely fill the coverslip without breaching the rims. Sample compression speeds between 100 µm/s and 1000 µm/s did not have any effect on object collection outcomes. In effect, the simple placement of coverslips on top the drop of sample by hand without a motorized translator was found to produce similar collection outcomes. Quantitative measurements confirmed that all the objects investigated were displaced and relocated at the rim regions to a very high degree.

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