Fluorescence microscopy living cell imaging

Taylor, D. L., and Wang, Y.-L. (eds.), 1989, Fluorescence Microscopy Living Cells in Culture. Part B Quantitative Fluorescence Microscopy—Imaging and Spectroscopy, Academic Press, New York, 503 pp. 

Xu X, Brzostowski JA, Jin T (2006) Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol Biol 346 281-96... 

Fluorescence microscopy techniques for live cell imaging... 

Fluorescence microscopy is one of the most powerful imaging methods in modern biomedical research. Noninvasive, fluorescence microscopy allows dynamic imaging of live cells, tissues, and whole organisms. This capability for live sample imaging, combined with a large repertoire of spectrally distinct fluorescent probes and a variety of biochemically specific labeling techniques, enables the direct visualization of complex molecular and cellular processes in real time under physiological conditions. 

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)...

Sako, Y Uyemura, T. Total internal reflection fluorescence microscopy for singlemolecule imaging in living cells. Cell. Struct. Funct. 2002, 27, 357-365. 

Tramier M, Coppey-Moisan M (2008) Fluorescence anisotropy imaging microscopy for homo-FRET in living cells. Methods Cell Biol 85 395-414... 

Elangovan, M., Day, R. N. and Periasamy, A. (2002). Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J. Microsc. 205, 3-14. 

Tramier, M., Zahid, M., Mevel, J. C., Masse, M. J. and Coppey-Moisan, M. (2006). Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microsc. Res. Tech. 69, 933-9. 

Pepperkok, R., Squire, A., Geley, S. and Bastiaens, P. I. (1999). Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr. Biol. 9, 269-72. 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

Sekar RB, Periasamy A (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160 629-33... 

Confocal fluorescence microscopy has been extensively used in cell biology. Single living cells can indeed be studied by this technique visualization of organelles, distribution of electrical potential, pH imaging, Ca2+ imaging, etc. (Lemasters, 1996). Interesting applications in chemistry have also been reported in the fields of colloids, liquid crystals and polymer blends. 

Lemasters J. J. (1996) Confocal Microscopy of Single Living Cells, in Wang X. F. and Herman B. (Eds), Fluorescence Imaging Spectroscopy and Microscopy, Chemical Analysis Series, Vol. 137, John Wiley ... 

There has been a continued interest in examining the properties of intact living cells using fluorescence microscopy. This field has seen considerable advances since the application of digital imaging techniques. In examining whole cells, one has to be especially aware of the location(s) of the probe. This is particularly important when bulk measurements are to be made on intact cells. 

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