Dataset Information

Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs.

ABSTRACT: A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.

SUBMITTER: Jurikova T

PROVIDER: S-EPMC7711807 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Bringing SEM and MSI Closer Than Ever Before: Visualizing <i>Aspergillus</i> and <i>Pseudomonas</i> Infection in the Rat Lungs.

Juříková Tereza T Luptáková Dominika D Kofroňová Olga O Škríba Anton A Novák Jiří J Marešová Helena H Palyzová Andrea A Petřík Miloš M Havlíček Vladimír V Benada Oldřich O

Journal of fungi (Basel, Switzerland) 20201030 4

A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dr ...[more]

PMID: 33143040

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Project description:BackgroundIn Italy the incidence of malignant mesothelioma (MM) among women is remarkably high, due to the several contexts in which women had been exposed to asbestos. However, very few studies in literature focus on the inorganic lung content in women. The aim of this retrospective, observational study is to investigate the asbestos lung burden, in terms of concentration, dimensions and type of asbestos, in 42 women who died from MM and had been non-occupationally exposed to asbestos during the activity of the asbestos-cement plant located in Broni (Pavia, Northern Italy) where mainly chrysotile, crocidolite and amosite were used.MethodsLung samples taken during forensic autopsies have been digested using sodium hypochlorite and filtered through a cellulose-ester membrane. The filter was examined using a scanning electron microscope and the chemical composition of the fibers was analyzed using an electron dispersive spectroscopy. The number of detected inorganic fibers, asbestos fibers and asbestos bodies (ABs) were normalized to 1 gram of dry tissue.ResultsIn six samples no asbestos has been detected. Overall, the most represented kind of asbestos was amosite, followed by crocidolite, tremolite/actinolite asbestos and chrysotile. The concentration of all inorganic fibers was significantly higher in women with environmental and household exposures compared with those with only environmental exposure (P=0.025), as well as the concentration of asbestos fibers (P=0.019) and ABs (P=0.049). We found a significant correlation between the concentration of asbestos fibers and the duration of exposure (rho =0.413, P=0.008), as well as with the latency of MM (rho =0.427, P=0.005). The distance of the residential address from the factory and the time spent daily in contact with asbestos did not influence the lung asbestos burden.ConclusionsThese results suggest the relevance of the lung clearance of asbestos, regarding mainly chrysotile. As a consequence, although scanning electron microscopy -energy dispersive X-ray spectroscopy (SEM-EDS) is considered the most reliable tool for assessing previous exposure to asbestos, its results should be interpreted with caution, especially in a legal context. In addition, our data confirm the relevance of environmental and household exposure in determining asbestos concentration in lungs and highlight the importance of household exposure.

Project description:BackgroundFilamentously growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. However, limited experimental throughput in bioprocess development still hampers taking advantage of their full potential. Miniaturization and automation are powerful tools to accelerate bioprocess development, but so far the application of such technologies has mainly been focused on non-filamentous systems. During cultivation, filamentous fungi can undergo remarkable morphological changes, creating challenging cultivation conditions. Depending on the process and product, only one specific state of morphology may be advantageous to achieve e.g. optimal productivity or yield. Different approaches to control morphology have been investigated, such as microparticle enhanced cultivation. However, the addition of solid microparticles impedes the optical measurements typically used by microbioreactor systems and thus alternatives are needed.ResultsAspergillus giganteus IfGB 0902 was used as a model system to develop a time-efficient and robust workflow allowing microscale cultivation with increased throughput. The effect of microtiter plate geometry, shaking frequency and medium additives (talc and calcium chloride) on homogeneity of culture morphology as well as reproducibility were analyzed via online biomass measurement, microscopic imaging and cell dry weight. While addition of talc severely affected online measurements, 2% (w v-1) calcium chloride was successfully applied to obtain a highly reproducible growth behavior with homogenous morphology. Furthermore, the influence of small amounts of complex components was investigated for the applied model strain. By correlation to cell dry weight, it could be shown that optical measurements are a suitable signal for biomass concentration. However, each correlation is only applicable for a specific set of cultivation parameters. These optimized conditions were used in micro as well as lab-scale bioreactor cultivation in order to verify the reproducibility and scalability of the setup.ConclusionA robust workflow for A. giganteus was developed, allowing for reproducible microscale cultivation with online monitoring, where calcium chloride is an useful alternative to microparticle enhanced cultivation in order to control the morphology. Independent of the cultivation volume, comparable phenotypes were observed in microtiter plates and in lab-scale bioreactor.

Dataset Information

Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs.

Publications

Bringing SEM and MSI Closer Than Ever Before: Visualizing <i>Aspergillus</i> and <i>Pseudomonas</i> Infection in the Rat Lungs.

Similar Datasets

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