Confocal fluorescence laser-scanning microscopy

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Quader H. Formation and disintegration of cistemae of the endoplasmic reticulum visuahsed in hve cells by conventional fluorescence and confocal laser scanning microscopy evidence for the involvement of Ca2+ and the cytoskeleton. Protoplasma 1990 155 166-175. 

Confocal laser scanning microscopy can be used in conjunction with microwave heating for examining the three-dimensional structure and cellular interrelationships in sections of paraffin-embedded tissues (Boon and Kok, 1994). Tissues are fixed with Kryofix, a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (PEG molecular weight 300) for 90 sec in a microwave oven. The use of thick paraffin sections (15 (xm) and fluorescently labeled antibodies is preferred. 

Meeuwissen, M.E.M.J., et al. 1998. A cross-section device to improve visualization of fluorescent probe penetration into the skin by confocal laser scanning microscopy. Pharm Res 15 352. 

The distribution of LCM in the brain parenchyma was further analyzed using fluorescently labeled LCM and confocal laser scanning microscopy. As in the case of unlabeled LCM, rats bearing 9L gliosarcoma tumors were injected intravenously (i.e., via tail vein) with diO-LCM and sacrificed 2 min later. The brains were processed as described elsewhere (ref. 531). In this case,... 

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.)...

TAT-peptide modified liposomes were prepared and cellular association and intracellular distribution of (double) fluorescently labelled particles were assessed by flow cytometry and confocal laser scanning microscopy. 

Fig. 2. Intracellular localization of TAT-liposomes. OVCAR-3 cells are incubated with 150 nmol of double fluorescently labelled TAT-liposomes for 1 h and subsequently incubated for 1 h (a) or 23 h (b) in liposomes-free medium. Thirty minutes before visualization the endocytic pathway is labelled with Lysotracker Red. Live cell imaging is performed with confocal laser scanning microscopy. Double labelled liposomes are used to study the integrity of the liposome during the uptake process co-localization of both liposomal labels would indicate that the liposomes are intact. 1 h both liposomal labels are localized at the plasma membrane, which represent intact cell-bound TAT-liposomes. The electronically merged image clearly shows lack of co-localization with the endocytic pathway marker, Lysotracker Red. This opposite to the 24 h incubation, both liposomal labels can be seen intracellularly in a punctuate pattern. In the electronically merged image, co-localization with Lysotracker Red is clearly visible. This indicates that the TAT-peptide modified liposomes bind to the plasma membrane and after internalization are present in endocytic vesicles. Therefore, we conclude that the liposomes are internalized by endocytosis. (Reproduced from (12) with permission from Elsevier Science)...

Fluorescent reporters allow detection in a single cell with epifluorescence and confocal laser scanning microscopy (Cormack et al., 1996 Tombolini et al., 1997 Phillips, 2001) or allow quantitative single-cell analysis with flow cytometry (Cormack et al., 1996 Valdivia and Ramakrishnan, 2000), enlarging their potential scope of use. 

Wolf, E. and Schiifller, A. (2005) Phycobiliprotein fluorescence of Nostoc punctiforme changes during the life cycle and chromatic adaptation characterization by spectral confocal laser scanning microscopy and spectral unmixing. Plant, Cell Environ., 28, 480-491. 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

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.

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