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The Emerging Role of Exosome Nanoparticles in Regenerative Medicine
Published in Harishkumar Madhyastha, Durgesh Nandini Chauhan, Nanopharmaceuticals in Regenerative Medicine, 2022
Zahra Sadat Hashemi, Mahlegha Ghavami, Saeed Khalili, Seyed Morteza Naghib
A pre-processing step is often necessary for biological sample preparation, to be visible under EM (Mehdizadeh et al. 2014). These sample preparations consisted of some treatments like dehydration (organic solvents are replaced by water), chemical fixation (chemical crosslinking of lipids with osmium tetroxide and proteins with formaldehyde or glutaraldehyde), and cryofixation (called cryogenic electron microscopy (cryo-EM)). Sometimes electron beam may damage the sample to overcome this problem. The cryo-EM could be applied for EVs analysis. It is the method used to study heterogeneous shapes of exosomes close to their native state. The specimen rapidly (in milliseconds) becomes cooled and frozen to cryogenic temperatures (usually at the temperature of liquid nitrogen), which are enough to form icecrystals. The water molecules are not regularly arranged in a hexagonal lattice and a vitreous ice is formed, which is an amorphous solid form of water (Tivol et al. 2008; Debenedetti 2003).
The Kinetics of Artificially Induced Membrane Fusion
Published in Sek Wen Hui, Freeze-Fracture Studies of Membranes, 1989
where Cij, Dij and fij denote the association (aggregation), dissociation, and fusion rate constants, respectively. These constants can be deduced from intermembrane forces, or measured experimentally by fusion assays.1 The time courses for membrane attachment and fusion between these vesicles or cells can then be calculated. Synchronized cryofixation experiments may then be performed to verify the process morphologically.
Lipid-Based Nanoparticles: SLN, NLC, and MAD
Published in Madhu Gupta, Durgesh Nandini Chauhan, Vikas Sharma, Nagendra Singh Chauhan, Novel Drug Delivery Systems for Phytoconstituents, 2020
Rita Cortesi, Paolo Mariani, Markus Drechsler, Elisabetta Esposito
At the end of the twentieth century the study of hydrated colloidal dispersions in fine vitreous ice slices was conducted using TEM (Fernández-Morán, 1960, Hutchinson et al., 1978). Particularly, such an approach became a common application after developing a technique for a rapid freeze of thin aqueous pellicules (Taylor and Glaeser, 1974, 1978). After the adjusting of this method, it was possible to routinely prepare aqueous dispersions as frozen-hydrated samples for the TEM visualization. In this way, a high-resolution cryogenic electron microscopy of vitrified samples of dispersions of colloids was obtained applying the thin-film technique (Adrian et al., 1984). The technical basics of cryofixation and its use in colloidal aqueous dispersions (Lepault et al., 1983, 1985) allowed the deep knowledge of structures and functions of colloids in pharmaceutical and biological fields (Cui et al., 2007, Dubochet, 2012, Dubochet et al., 1985, Dubochet and Adrian, 1988). Thus, in order to obtain good results, cryogenic TEM has to follow some rules for preparing thin films of frozen-hydrated samples (Adrian et al., 1984). For example, to obtain a slim film of particles in a single layer passing through the laced carbon backing film holes, direct and complete contact with an absorbent filter paper is required to absorb and remove the material in excess. Afterwards the cryofixation has to be obtained by quick immersion of the sample into the cryogen (i.e., liquid ethane). For further transfer to TEM, the obtained specimens should be maintained at low temperatures. This condition, aimed to prevent the recrystallization of vitrified samples, is allowed with the use of supports for low temperatures cooled with liquid nitrogen. Moreover, due to the environmental moisture, the possible presence of frozen water over the top of thin film must be avoided using appropriate breechblock systems. Furthermore, in the TEM analyses, unfixed aqueous colloidal systems constituted of hydrocarbons display weak amplitude contrast and are sensitive to the electron beam radiation damages. Thus to increase the poor contrast and to detect specific details of the sample, TEM analyses must be conducted using conditions of electron beam low dose, optimal defocus, and/or a combined apparatus between an energy filtering system and phase plate.
Mass spectrometry-based phospholipid imaging: methods and findings
Published in Expert Review of Proteomics, 2020
Al Mamun, Ariful Islam, Fumihiro Eto, Tomohito Sato, Tomoaki Kahyo, Mitsutoshi Setou
PLs imaging at the cellular level has been made possible by SIMS imaging as it offers the capability of resolving very small features which can be as low as a hundred nanometers. Sample for single-cell analysis includes the isolated cells from the specimen or cultured cell. Unfortunately, live cells cannot be analyzed directly in SIMS as it works under a high vacuum condition. Generally, cells are extracted or grown on an appropriate substrate such as silicon wafer or gold-coated silicon wafer [58] followed by fixation to minimize sample degradation. Two fixation methods are commonly applied: (i) chemical fixation using glutaraldehyde, and (ii) cryofixation. A cryofixation method, namely plunge fixation, has been shown to be advantageous for single-cell lipid imaging [59]. In plunge freezing, samples are stored in liquid nitrogen at −196°C after washing by a mixture of propane and isopentane (3:1). Interestingly, enhanced signal intensity for PLs has been reported when matrix solution is added on the cell surface prior to the fixation [58]. After fixation, frozen samples are freeze-dried and stored until analysis.
Use of electron microscopy to study megakaryocytes
Published in Platelets, 2020
Cyril Scandola, Mathieu Erhardt, Jean-Yves Rinckel, Fabienne Proamer, Christian Gachet, Anita Eckly
MKs (from bone marrow (BM) or cultures) are fixed in glutaraldehyde, post-fixed in osmium, dehydrated with alcohol and embedded in a plastic resin. The samples are sectioned (80–100 nm), counterstained with heavy metals and examined under a transmission electron microscope at 80–120 kV [24]. An alternative method to chemical fixation is cryofixation. Taking into consideration the size of the sample, electron microscopists have a wide range of cryo-techniques at their disposal. Thin sample, such as platelets, can be vitrified in a thin layer of ice on a TEM grid by plunge freezing. Vitrification of larger cells up to ~ 200 µm thickness such as MKs can be achieved by high-pressure freezing (HPF) by combining jets of liquid nitrogen simultaneously with very high pressures to prevent the formation of ice crystals. Significant advantages of cryoimmobilisation over chemical fixation is the preservation of the molecular structures in their native environment and the possibility to catch a dynamic cellular process [25].
Still challenging: the ecological function of the cyanobacterial toxin microcystin – What we know so far
Published in Toxin Reviews, 2018
Azam Omidi, Maranda Esterhuizen-Londt, Stephan Pflugmacher
The hypothesized involvement of MCs in photosynthesis was further supported by studies using an immunogold-labeling technique that disclosed that the thylakoids membrane is the most MC-occupied cell site followed by the nucleoplasmic area. Physically, more than two-thirds of MCs were attached to the thylakoids membranes (Shi et al., 1995; Young et al., 2005,2008). Although a further study using the cryofixation/cryosectioning technique demonstrated that most of the MCs were localized in the nucleoplasmic area and intracellular inclusions such as carboxysomes and polyphosphate bodies, rather than thylakoids membranes and the cell wall (Gerbersdorf, 2006). Under high light irradiation, the ratio of MCs in outer to inner cellular parts increased, and a higher percentage of MCs were found close to the thylakoids membrane suggesting the probable role of MCs in light adaptation (Gerbersdorf, 2006). Moreover, the M. aeruginosa mcyA-knockout mutant has been found to be dominant under low light, with the toxic genotype M. aeruginosa PCC 7806 showing a greater fitness to high light suggesting that MCs play a role in protection against photooxidation (Phelan & Downing, 2011). In contrast, another study showed that under both low and high light irradiation (1480 and 5920 lm m−2, respectively) a mixed culture was dominated by the MC-producing strain M. aeruginosa UTCC 300 which further emphasized the importance of MCs in light adaptation (Renaud et al., 2011). Comparative proteomic studies also revealed two NADPH-dependent reductases, phycobiliproteins, and RuBisCo, which is a Calvin cycle enzyme, were expressed differently in the wild-type and mcyB− mutant of M. aeruginosa PCC 7806. Furthermore, MC-protein binding was significantly enhanced under high light (51 800 lm m−2) which was assumed to increase the protein stability and avoid redox changes (Zilliges et al., 2011). Therefore, the potential role of MCs in photooxidative protection under high light is an advantage for the organism (Gerbersdorf, 2006; Phelan & Downing, 2011). In another study with M. aeruginosa PCC7806 and its MC-deficient mutant, differences in metabolic responses between strains upon exposing to high light intensity (18 500 lm m−2) were observed. Trehalose and sucrose, two general stress markers, accumulated more in the mutant while carbon reserves such as glycolate accumulated faster in the wild type. Additionally, the photosynthesis rate and high molecular weight carbohydrate contents were greater in the wild type (Meissner et al., 2015).