PhD week 6: Laser Scanning Confocal Microscopy

Bone cells viewed by laser confocal microscopy. Courtesy of the Wellcome Images photostream. License: CC: BY-NC-ND

This week, I had the opportunity to learn more about laser scanning confocal microscopy (LSCM). This type of microscopy uses lasers to excite fluorescent compounds in a very thin layer of the sample, allowing very clear images to be produced. Images can be made at a ranges of depths in the sample, and can then be merged together to give an idea of the 3D structure of the subject being imaged, such as the proturan leg shown in this video.

While it is frequently held that chitin can fluoresce to a degree, it appears that it is rather the presence of proteins and other sclerotizing agents in the cuticle that is primarily responsible for any autofluorescence seen. In the celebrated case of scorpions, it is believed that an alkaloid and a coumarin compound causes the fluorescence, while in froghoppers, it is resilin.

If the chitin is not fluorescing adequately, there are a number of stains that can be used. Eosin Y was judged to be most useful of a number of stains in one study, however in the test I did this week, I judged that it primarily stained the membranes, and not so much the sclerotised structures. A few other stains that may or may not prove useful include calcofluor, primulin and Congo red.

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Read:
Koerner L, Gorb SN, Betz O. 2012. Functional morphology and adhesive performance of the stick-capture apparatus of the rove beetles Stenus spp. (Coleoptera, Staphylinidae). Zoology 115: 117-127

Grant PR, Grant BR. 2008. How and Why Species Multiply. The Radiation of Darwin's Finches. Princeton: Princeton University Press

McCulloch D. 2010. A History of Christianity: The First Three Thousand Years London: Penguin

Psalms 37–38

Websites:
New Zealand Tango Festival, 19–26 June 2012

OsiriX an open-source volume rendering program.

Suraj Gupta—How R Searches and Finds Stuff

Steveko's Blog—10 things I hate about Git

BeetleBase and FlyBase genomic databases.

Installing Arial fonts in Wine

WineHQ information about Leica LAS AF Lite

Volume 3 of the NZ Inventory of Biodiversity published

Listened:
Demon Hunter—45 Days

Zao—Awake?

Foo Fighters—Wasting Light

Watched:
Star Trek: Deep Space Nine Season 3

Further Seems Forever—Light Up Ahead music video