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Technological Developments in the Study of the Microcirculation
Published in John H. Barker, Gary L. Anderson, Michael D. Menger, Clinically Applied Microcirculation Research, 2019
Marcos Intaglietta, Konrad Messmer
In many tissues, illumination can only be obtained by incident light, which poses an additional limitation on contrast and resolution of the moving red blood cells. This problem has been in part resolved by the use of fluorescent markers that stain either the red blood cells or the plasma and therefore enhance contrast and provide more distinct optical signatures for the passage of red blood cells. Epifluorescence microscopy is shown to underestimate the actual velocity by at most 20%.25
Bacteriocins as Anticancer Peptides: A Biophysical Approach
Published in Ananda M. Chakrabarty, Arsénio M. Fialho, Microbial Infections and Cancer Therapy, 2019
Filipa D. Oliveira, Miguel A.R.B. Castanho, Diana Gaspar
Different studies show the importance of microscopy techniques for detailing bacteriocins’ mechanism(s) of action. Chen et al. relied on confocal microscopy to investigate the distribution of KL15 in SW480 cells [96]. Confocal microscopy allows imaging fixed or living cells and tissues previously labeled with fluorescent probes, with an increased lateral and axial resolution when compared to epifluorescence microscopy [113]. In this study, the bacteriocin-derived peptide KL15 was labeled with iV-hydroxysuccinimide (NHS)-fluorescein, a green fluorescent dye, while the cell membrane was labeled with the red fluorescent dye di-8-ANEPPS and the blue fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) was applied to label the cell nucleus [96]. Evaluating the colocalization of the dyes it was possible to infer KL15 localization inside the cell. Confocal microscopy images indicated that KL15 enters the cells and causes significant changes in their morphology [96]. Nonetheless, red fluorescence was detected and found to be colocalized with DAPI, used for nuclei staining. This observation was in agreement with the results obtained from SEM, as it was demonstrated that KL15 may damage treated cells by cell membrane penetration and consequent intrusion into the cells [96].
Cyclospora cayetanensis: Portrait of an Intriguing and Enigmatic Protistan Parasite
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Annunziata Giangaspero, Robin B. Gasser
In a recent study on ready-to-eat salads, samples were processed as described by Giangaspero et al. [57] and Dixon et al. [118], but the methods were slightly modified. In brief, mixed salads were weighed and washed in 200 mL of buffered detergent solution (containing phosphate-buffered saline 10X [PBS], 0.1% Tween-80, 0.1% sodium dodecyl sulphate [SDS] and 0.05% antifoam B emulsion) using an orbital shaker for 15 min at 120 rpm. All of the solutions were centrifuged, then the final pellets were resuspended in 3 mL of buffered detergent solution, and pooled into one centrifuge tube. The pooled tube was centrifuged, resuspended in 2 mL of buffered detergent solution before microscopy and molecular investigation [119]. Concentrated samples can be examined by bright field microscopy or DIC, followed by epifluorescence microscopy. However, none of these methods is specific for Cyclospora, so that molecular tools should be used to confirm results, and may provide additional information.
Injectable calcium phosphate scaffold with iron oxide nanoparticles to enhance osteogenesis via dental pulp stem cells
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Yang Xia, Huimin Chen, Feimin Zhang, Lin Wang, Bo Chen, Mark A. Reynolds, Junqing Ma, Abraham Schneider, Ning Gu, Hockin H. K. Xu
Cellular imaging on CPC after seeding was performed by immersing the scaffold in a live/dead staining solution (Invitrogen, Carlsbad, CA, USA). Cells were examined via epifluorescence microscopy (Eclipse TE-2000 S, Nikon, Tokyo, Japan). Three images were taken at random locations for each sample, with six samples yielding 18 images for each group at each time point. The images were analysed by Image-Pro Plus 6.0 software. Live cell spreading area was calculated as: S = Stotal/NLive, where Stotal is the total cell spreading area in the image, NLive is the number of live cells [39]. A cell counting kit (CCK-8, Enzo Biochem, New York, NY, USA) was used to evaluate the adhered cell ratio normalized by culture well control at 4 h after seeding. After incubating with 10% CCK-8/media in the dark for 2 h, optical density (OD) was read at a wavelength of 450 nm. Cell adhesion ratio = OD value of scaffold group/OD value of the culture well control [31].
Zosteric acid and salicylic acid bound to a low density polyethylene surface successfully control bacterial biofilm formation
Published in Biofouling, 2018
C. Cattò, G. James, F. Villa, S. Villa, F. Cappitelli
Epifluorescence microscopy was additionally used to provide image analysis and in situ quantification of bacterial cells. Figure 1 shows direct microscopic visualizations of the total biofilm biomass on functionalized and non-functionalized coupons and stained for live and dead cells. Microscope assay revealed that differences in dead cell data were not statistically relevant (Table 1 and Figure 1A). Conversely, biofilms on LDPE-CA and LDPE-SA showed a significant decrease in the number of live cells, corresponding to 56.7 ± 11% and 70.6 ± 7.3% in respect to the LDPE control sample (Table 1, Figure 1B and C), confirming the results obtained in the plate count viability assay. No significant differences were detected in the live cell data between the LDPE and the LDPE-OH and LDPE-COOH linker samples (Table 1).
In vitro assessment of inter-kingdom biofilm formation by bacteria and filamentous fungi isolated from a drinking water distribution system
Published in Biofouling, 2019
Tiago Barros Afonso, Lúcia Chaves Simões, Nelson Lima
Epifluorescence microscopy is commonly used to obtain rapid, inexpensive qualitative information on fungal cells (Ahmad and Khan 2012). CSLM has the advantage of allowing 3-D characterization of the biofilm structure, but it is much more expensive than fluorescence microscopy (Simões et al. 2015; Schlafer and Meyer 2017). Consequently, epifluorescence microscopy was used as a first approach to qualitatively analyse inter-kingdom biofilm development over time. CW staining allows the visualization of fungal cell walls because of its affinity for β(1-3) and β(1-4) polysaccharides in cellulose, carboxylated polysaccharides, and chitin (Siqueira et al. 2011, 2013; Simões et al. 2015). DAPI was selected due to its frequent use in microscopy as a nuclear counterstain to assist in morphological observations of the bacteria (Kormas et al. 2010; Siqueira et al. 2013). The excitation wavelength for CW and DAPI was the same and both signals were blue; however, they were distinctive in colour intensity and brightness in microscopic observations. In inter-kingdom biofilms, the stronger fluorescent signal coming from the fungal staining by CW made the acquisition of clear images showing both organisms more difficult. This happened especially when germinated spores were used as the starting inoculum due to the higher number of fungal hyphae (data not shown). Besides the difference in fluorescence intensity between microorganisms, to visualize bacteria it was usually necessary to focus on a different focal plane, usually lower, which made obtaining inter-kingdom biofilm images more difficult. Despite these drawbacks, the use of these stains allowed the discrimination of the microorganisms in inter-kingdom biofilms.