Confocal Microscopy Lab

explanatory Essay
861 words
861 words

Confocal Microscopy Lab

Confocal microscopy uses a laser that produces excitation light. This laser light reflects off of a dichroic mirror and then the laser light hits two mirrors that are mounted on motors. The mirrors then scan the laser light across the sample. Dye that is found in the sample then fluoresces (Weeks, 2003). Filamin was labeled with a red fluorescent label rhodamine (TRITC) and actin was labeled with the green fluorescent label fluoroscein (FITC) which was conjugated to the actin-binding fungal toxin phalloidin. The emitted light from the dyes passes back through the mirrors and passes through the dichroic mirror and is focuses into a pinhole. With confocal microscopy, a complete image of the sample is never seen. Only one point of the sample can be observed at a time. The amount of light that passes back through is detected by the microscope (Ladic, 1995). The intensity of the red light seen is proportional to the amount of filamin present and the intensity of the green light seen is proportional to the amount of actin present in the sample of Drosophila melanogaster ovaries.

Confocal microscopy is able to produce images that are very free from interference. The confocal pinhole allows the microscope to reject out of focus fluorescent light (Weeks, 2003). This means that the image comes from a thin section of the ovary sample. Many thin sections will be scanned through the sample; this allows a clean three dimensional image to be made. A confocal microscope has a few advantages over regular optical microscopes. Confocal microscopes have controllable depth of field, the elimination of image degrading information that is out of focus, and the ability to collect series of data from s...

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.../ladic/overview.html. Accessed 6 December 2004.

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Robinson, D.N, T.A. Smith-Leiker, N.S. Sokol, A.M. Hudson and L. Cooley. 1997. “Formation of the Drosophila ovarian ring canal inner rim depends on cheerio.” Genetics. 145, 1063-1072

Shilling, Kristen (David S. Richard). “Ovarian nurse cell ring canal formation in wild-

type and insulin signaling mutant female Drosophila melanogaster.”

Tilney, L.G., M.S. Tilney, and G.M. Guild. 1996. “Formation of actin filament bundles in the ring canals of developing Drosophila follicles.” The Journal of Cell Biology. 133, 61-74.

Weeks, Eric. 2003. “How does a confocal microscope work?” Physics Department, Emory University. Accessed 3 December 2004

In this essay, the author

  • Explains that confocal microscopy uses a laser that produces excitation light. the laser light reflects off of the dichroic mirror and then hits two mirrors that are mounted on motors.
  • Explains the advantages of a confocal microscope over regular optical microscopes, such as controllable depth of field, elimination of image degrading information, and the ability to collect series of data from thick samples.
  • Explains that oocyte development in drosophila proceeds through fourteen stages. the first seven stages are pre-vitellogenic, and the last seven are vitellogenic.
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