Fluorescence microscopy double labeling

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)...

Figure 11.2 Subcellular distribution of fluorescein-labeled DNA fragments and plasmid DNA following microinjection into the cytoplasm. Double stranded circular plasmid DNA (3 kb and 6 kb) and DNA fragments (20,100,250 and 1 kb) were covalently labeled with fluorescein and microinjected into the cytoplasm of adherent HeLa cells as described in Lukacs et al., 2000. Following micro injection, cells were either fixed or incubated for 45 minutes at 37 °C and the distribution of DNA was visualized by fluorescence microscopy, (see Color Plate 10)...

Figure 21. Six sequential images of the same sample area of individual carbocyanine dye molecules spread over a polymethylmethacrylate (PMMA) film as recorded by scanning near field optical microscopy. The image dimensions are 2.3 x 2.7-/nn cut out from 4 x 4-fim records of 256 x 256 data points. The excitation polarization was random in (A-D) and linear along y and x, respectively, in ( ) and (F). The emission polarization was measured along y and x in (B) and (C) and not otherwise. Some fluorescence spots are labeled for discussion in the text. The various shapes of the fluorescence peaks (circular spots, rings, arcs, and double arcs) are striking. These shapes can be explained by molecular dipoles being excited by the inhomogeneous electric field at the aperture. (Adopted from [83].)...

External coalescence leads to release of internal droplets and their contents to the outer continuous phase. The release of a fluorescent label from the internal aqueous phase of a water/oil/water double emulsion was visualized with fluorescence microscopy and indicated the occurrence of external coalescence (Figure 3.16) (Gonz ez-Ochoa et al., 2(X)3). External coalescence was directly detected in bulk water/oil/water systems (see Figure 3.17) (Pays et al.,... 

N. L. Izatt, J. A. Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy. Photochem. Photobiol. 2000, 71, 634-639. 

Exemplificative results of double IMF experiments for the localization of autophagy markers (beclin-1 and LC3B) in Purkinje neurons labeled with the anti-calbindin antibody are shown in Fig. 5. Micrographs are taken with conventional fluorescence microscopy. Results of quantitative analysis are reported in Fig. 6. 

Fig. 10 Confocal microscopy double IMF of neuroblastoma B50 cells. Staining for mitochondria green) and lysosomes (red) in (a) controls, (b) after 48-h treatment with cisPt, and (c) recovery. Images show that, after cisPt, lysosomes and mitochondria are distributed in cytoplasmic clusters compared to control, but do not show colocalization. During recovery, cells assume a morphology similar to control cells and show an increase of lysosomes. Immunohistochemistry for LC3B (green) and lysosomes (red) in (d) control and (e) recovery cells. Graph analysis shows the fluorescence peak and colocalization of the two labels. DNA is counterstained with Hoechst 33258 (blue). Scale bars 20 pm...

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. 

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