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Medical and Biological Applications of Low Energy Accelerators
Published in Vlado Valković, Low Energy Particle Accelerator-Based Technologies and Their Applications, 2022
The live-cell imaging facility at SNAKE is now able to routinely perform ambitious experiments. In addition to recording time series after single ion irradiation in defined patterns (Hable et al. 2012), experiments with selective high dose irradiation of cells within a reasonable time span can be performed. This raises the possibility to define the minimal dose needed for DNA repair proteins to accumulate in visible foci. Further, cellular substructures like nucleoli or different chromatin states (heterochromatin vs. euchromatin) can be targeted and irradiated with a defined number of ions, e.g., to investigate differential response mechanisms. Now, the setup is modified to perform three-colour LCI experiments. This enables the study the behaviour of three different proteins tagged with different fluorescent markers.
Identifying Nanotoxicity at the Cellular Level Using Electron Microscopy
Published in Suresh C. Pillai, Yvonne Lang, Toxicity of Nanomaterials, 2019
Kerry Thompson, Alanna Stanley, Emma McDermott, Alexander Black, Peter Dockery
The microscopic approach results in not only nanoparticle detection but also precise localisation in tissues, cells, and intracellular compartments. This precision has resulted in greater insight into the pathways involved in their interactions with living systems. Quantum dots, fluorescent dyes, and nanoparticle conjugates have been used to visualise nanoparticle cell dynamics by light microscopy and confocal microscopy. Flow cytometry has been employed to track cellular uptake (Elsaesser et al., 2010). The key advantage of adopting light microscopical approaches is that they can permit live cell imaging and analysis in a relatively quick and easy manner in terms of sample preparation with the significant benefit of not being sample destructive. The main disadvantage is that there is no resolution of nanoparticles therefore no absolute quantification and no imaging of bare particles. The recent emergence of super-resolution light microscopy techniques may provide sufficient resolution at the light microscopic level, promising a potential tool for dynamic in vivo studies (Owen et al., 2013).
Rabies Virus Neurovirulence
Published in Sunit K. Singh, Daniel Růžek, Neuroviral Infections, 2013
Claire L. Jeffries, Ashley C. Banyard, Derek M. Healy, Daniel L. Horton, Nicholas Johnson, Anthony R. Fooks
The use of lyssaviruses as transneuronal tracers in future studies will allow further aspects of rabies neurovirulence and pathogenesis to be investigated and understood (Ugolini 2008). Further study and modeling of the virus-host interactions of both rabies and other neurotropic viral infections will help to clarify the subversion and avoidance mechanisms the virus uses to circumnavigate the hosts defenses. The continued use of technologies such as live-cell imaging in the future will enable the dynamics and spatiotemporal relationships in these interactions to be further analyzed (Chevalier et al. 2010). The development of safe, efficacious and affordable tools for preexposure and postexposure prophylaxis with simpler, single immunization regimens could lead to the elimination of rabies from areas of economic and political instability (Faber et al. 2009). Live-attenuated vaccines are also potential future candidates for treatment in the early stages of clinical human rabies as they are capable of inducing effective immune responses to clear virulent rabies virus from the CNS (Faber et al. 2009).
Chaetocin induced chromatin condensation: effect on DNA repair signaling and survival
Published in International Journal of Radiation Biology, 2021
A. Sak, K. Bannik, M. Groneberg, M. Stuschke
Second, to preclude preferential death of CICC cells which may falsify the results regarding reversibility, live cell imaging was performed to monitor the fate of the cells. For this purpose, chaetocin treated cells were stained with Hoechst 33342 and monitored in real time up to 16 h after washout. The data showed that about 33.5% ± 9.9% of the cells with CICC phenotype were reversible. For a more detailed quantification, the chaetocin treated cells were divided in cells without CICC (normal) and cells with CICC phenotype, either reversible or nonreversible. These cells were then characterized by Hoechst 33342 staining with respect to apoptotic cell death. The results showed that only 18% ± 6% of CICC reversible cells died. Sham treated cells did not show any apoptotic features during the observation time.
Characterizing the DNA damage response in fibrosarcoma stem cells by in-situ cell tracking
Published in International Journal of Radiation Biology, 2019
Qibin Fu, Jing Wang, Tuchen Huang
Live-cell imaging was adopted in this study. In order to do a comparative study, cells were synchronized at G1 phase. One reason was to ensure that the DNA contents and cell cycle distribution were similar between CSCs and non-CSCs. Another reason was to consider the repair kinetics. Deckbar et al. reported that G1/S checkpoint is slowly activated. The G2/M checkpoint, in contrast, is quickly activated but only responds to a level of 10–20 DSBs such that cells with a low number of DSBs do not initiate the checkpoint (Deckbar et al. 2011). By choosing IRIF number, area and fluorescence intensity as endpoints, we found that CSCs had fewer IRIF number and smaller IRIF size compared with HT1080 cells (Figure 2). Since the dose response of 53BP1 IRIF depended on the cell type, radiation dose, incubation or time after irradiation and the duration of cell cycle (Marková et al. 2007; Belyaev 2010), actual kinetics of DSBs did not always coincide with the kinetics of 53BP1 IRIF. Even so, several papers reported that the number of 53BP1 IRIF is closely associated with the number of DNA DSBs (Schultz et al. 2000; Rappold et al. 2001). Furthermore, larger IRIF is more persistent and are closely correlated with cell lethality (Banáth et al. 2010).
Measuring and interpreting platelet-leukocyte aggregates
Published in Platelets, 2018
Michaela Finsterbusch, Waltraud C. Schrottmaier, Julia B. Kral-Pointner, Manuel Salzmann, Alice Assinger
Live cell imaging largely relies on fluorescent tools, including probes, antibodies, biosensors or reporter mice for visualising cells and/or structures without interfering with their phenotype and function. Early epifluorescence IVM studies primarily utilised intravascular administration of nonspecific cell dyes (e.g. rhodamine 6G), or ex vivo labelling and adoptive transfer of platelets into donor animals (58,62–64). However, the lack of clear distinctions between specific cell types using unspecific dyes limits the analysis of PLAs. Also, ex vivo staining and transfusion of purified platelets does not reflect the in vivo situation as it does not allow for analysis of unlabelled endogenous platelets and further harbours the risk of platelet activation due to in vitro handling.