Confocal fluorescence laser-scanning microscopy staining

Fig. 13.3. This figure demonstrates the distribution of fluorescently tagged LCM in brain parenchyma analyzed by confocal laser scanning microscopy. Rats bearing 9L tumors were administered diO-LCM and sacrificed 2 minutes later. Vibratome sections were counterstained with TR-WGA which binds to tumor cells and distinguishes the tumor area from the surrounding normal tissue. Comparison of the area stained with TR-WGA (tumor cells) (B) and that stained with diO (A) indicates that LCM were associated with a large portion of the tumor. (Taken from ref. 531.)...

Fig. 4. Combined use of flow cytometry/cell sorting and confocal laser scanning microscopy. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine and then analyzed for mitochondrial membrane potential and NAD(P)H fluorescence (A). Cells in different regions of the cytogram were then sorted, and subsequently stained with the DNA fluorochrome Hoechst 33342. These cells were then examined by CLSM. (B), (C), and (D) show three-dimensional reconstructions of nuclei from cells sorted from healthy, early apoptotic, and late apoptotic populations. Whereas healthy cells show normal round nuclei, early and late apoptotic cells show progressive chromatin condensation/margination and nuclear fragmentation.

Figure 4. Subcellular localization of the photosensitizer BPD-MA. OVCAR-5 cells were incubated in 92 nM BPD-MA for 3 h and 10 nM rhodamine 123, a mitochondrial probe, for 20 min. Imaging was performed using confocal laser scanning microscopy (CLSM). (A) Exclusively mitochondrial green fluorescence of rhodamine 123 (B) red BPD-MA fluorescence (C) overlay of A -i- B, where yellow indicates co-localization (D) DIC transmission image. The colocalization in (C) indicates that BPD-MA localizes to mitochondria, but also stains other subcellular structures.

Due to the cationic nature of chitosan, it can bind with negatively charged DNA by electrostatic interaction, which can be determined by competitive binding tests using ethidium bromide (EtBr) as a DNA stain [54]. As chitosan is added to a solution of EtBr-stained DNA, cationic chitosan binds with DNA which causes a decrease in fluorescence, determined by confocal laser scanning microscopy [55]. There are various methods for the determination of the extent of DNA complexation or encapsulation into chitosan nanoparticles, such as gel electrophoresis [54], Pico Green assay [55], and photoelectric methods. 

Quantifying Biofilm Parameters As the amount of biofilm biomass is proportional to current production, quantifying biomass is important to assess BES performance. The anode biofilms can be detached from the electrode with a spatula or razor, and the biofilm biomass can be calculated from the total cell protein [21]. Alternatively, the cells can be stained with fluorescence dyes such as the BacLight viability kit (Invitrogen) and examined by Confocal Laser Scanning Microscopy (CLSM). The... 

Fig. 8.7 Fluorescence in situ Hybridisation (FISH) overlay image of microbial aggregate involved in anaerobic methane oxidation. Different microbes were stained using different oligonucleotide probes. Core is ANNM-2 Archaea, surrounded by sulphate-reducing Desulfosarcina imaged by laser scanning confocal microscopy. From V.J. Orphan etal. in Proc. Natl. Acad. Set USA, Vol. 99, 7663—7668, 2002. Reproduced with permission. Copyright National Academy of Sciences (2002).

Confocal scanning laser microscopy has been combined with the use of fluorescent stains to measure the distribution and concentration of specific analytes within biological tissue. Stains are used to bind selectively with the analyte under study, and the correct laser beam is selected to excite the stain used to give a fluorescent signal. The technique can also be... 

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