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Confocal - Microscopy Imaging Techniques All about Microscopy, Confocal Microscopy, Fluorescence microscopy and Other Imaging Techniques


Confocal Microscopy Exam Question -- Please Help

Confocal Microscopy Exam Question -- Please Help - Confocal - Microscopy Imaging Techniques

Confocal Microscopy Exam Question -- Please Help - All about Microscopy, Confocal Microscopy, Fluorescence microscopy and Other Imaging Techniques


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Old 04-24-2010, 03:53 PM
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Default Confocal Microscopy Exam Question -- Please Help



So I just had my final exam for Cell Biology and this was one of the questions on it I can't figure out, hoping someone may be able to shed some light/clear up the issues I have. This is my first cell biology course (2nd year university)

Calcimycin, a toxin produced by the bacterium Streptomyces chartreusensis, acts as a mobile ion-carrier that allows Ca2+ ions to freely cross eukaryotic cell membranes, and indictors like Fluo5N give off fluorescence when bound to these ions. When Fluo5N is introduced into the same kind of yeast cells we used in our discussion of the two-hybrid analysis, and these cells are then treated with calcimycin, what organelle would you expect to see fluoresce when the cells are examined by confocal microscopy?

A) Mitochondria
B) Lysosome
C) Proteasome
D) RER
E) None of the Above

Alot of things jump out at me right away. Firstly, I must mention we've heard nothing of the toxin nor the fluorophore in class, upon further research on the internet I've found that Calcimycin (aka 823187) does indeed bind and inhibit Mitochondria.

When looking at this question, it reminded me strongly of a previous year's exam question which substituted yeast for bacteria (no organelles) and I thought this might be the way the professor was trying to trick us. I would have (initially) thought that all organelles would fluoresce equally as the toxin would introduce Calcium into all parts of the cell.

I know that bacterial cell walls allow for generalized passage of ions (e.g. calcium or Fluo5N) but are fungal walls significantly different in their function. I would assume their walls would allow for generalized passage as well or else unnecessary energy would be wasted.

Also the mention of "the same kind of yeast cells used in our discussion of yeast 2-hybrid" is confusing as well. The only thing I can think of there is that the yeast are deficient in three amino acids (2 are plasmid markers, the third is a result of activation of the fish-bait gene). I don't think this is what he meant, moreso that the cells were susceptible to plasmid uptake (predominantly - charged particles = similar to Fluo5N maybe)

In actuality, I picked mitochondria not because I knew it was inhibited, but because the three other organelles contain inner exoplasmic face vs a mitochondrial double membrane causing an inner cytosolic face (seemed like the only relative difference). However, in retrospect this may create difficulty for the Fluo5N to cross both membranes.

P.S.
A few things I've found out since then. Fluo-5n is usually introduced via an ester, due to its negative charge (COOH) preventing membrane crossing. If the ester was to cross into the intermembrane space of the mitochondria I would assume it would become hydrolyzed and remain there (technically not in the mitochondria?) Also Fluo-5n does not have as high an affinity for calcium than other fluorophores, this makes me think a relatively high positive charge (ie. low pH) environments (mitochondria/active lysosome) would not favour calcium gradient flow enough to warrant confocal detection.

Thanks in Advance!
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