Video microscopy and confocal laser scan microscopy to study the mechanisms of cytotoxicity in individual living cells. Application and new developments

2002 ◽  
Vol 55 (S1) ◽  
pp. S25-S29
Author(s):  
J. F. Nagelkerke ◽  
H. J. G. M. de Bont
Keyword(s):  
Living Cells ◽  
Video Microscopy ◽  
Confocal Laser ◽  
New Developments ◽  
Confocal Laser Scan Microscopy

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

Expression of the gap junction proteins connexin31 and connexin43 correlates with communication compartments in extraembryonic tissues and in the gastrulating mouse embryo, respectively

1996 ◽  
Vol 109 (1) ◽  
pp. 191-197
Author(s):  
E. Dahl ◽  
E. Winterhager ◽  
B. Reuss ◽  
O. Traub ◽  
A. Butterweck ◽  
...  
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Confocal Laser ◽  
Mouse Development ◽  
Confocal Laser Scan Microscopy ◽  
Early Mouse ◽  
Junction Proteins ◽  
In Cells ◽  
Extraembryonic Tissues

We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.

scholarly journals Nuclear halo from Bradysia hygida (Diptera:Sciaridae) salivary gland polytene cells

2005 ◽  
Vol 48 (3) ◽  
pp. 351-357 ◽  
Author(s):  
Celso Aparecido Polinarski ◽  
José Luis da Conceição Silva ◽  
Liya Regina Mikami ◽  
Maria Aparecida Fernandez
Keyword(s):  
Salivary Gland ◽  
Structure And Function ◽  
Confocal Laser ◽  
Work Analysis ◽  
Confocal Laser Scan Microscopy ◽  
Nuclear Halo ◽  
Hypotonic Buffer ◽  
And Function ◽  
Polytene Nuclei ◽  
Bradysia Hygida

A protocol for recovered nuclear halos from insect polytene nuclei after the extraction of the nuclear proteins using LIS detergent is reported in this work. Analysis was carried out using fluorescence and confocal laser scan microscopy. The extraction of nuclear halos was possible only with nuclei-fraction isolation in hypotonic buffer without spermine and spermidine. The recovered nuclear halos from Bradysia hygida salivary gland polytene nuclei, contributed greatly to the study of the structure and function of these special organelles.

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