Thesis-PDF - IAP/TU Wien
Thesis-PDF - IAP/TU Wien
Thesis-PDF - IAP/TU Wien
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Cell parts can then be collected and purified. The disrupted rigid outer cell<br />
walls can be eliminated in another cycle of centrifugation and washing processes.<br />
After a series of purification steps a solution of crystalline cell parts containing<br />
only a minor fraction of dissolved biological material is obtained. Dependent on<br />
the various forms of treatment before the explosion, the results obtained can differ<br />
to a great extent. Variations of this procedure included tests on cells treated with<br />
detergent, non-treated live cells and fixated 2 cells.<br />
5.2 Experimental Setups<br />
5.2.1 Optical Microscopy and Fluorescence Microscopy<br />
In Pisa optical and fluorescence microscopy were carried out with a Zeiss Axioplan<br />
fluorescence microscope equipped with an epifluorescence system, various<br />
magnifications and a 100W mercury lamp.<br />
The following filter combination could be used: a UV-blue set (an 8 nm bandpass<br />
excitation filter, 365 nm; chromatic beam splitter, 395 nm; barrier filter,<br />
397 nm; 800 µW/cm 2 ) and a blue-violet set (an 8 nm bandpass excitation filter,<br />
436 nm; chromatic beam splitter, 460 nm; barrier filter, 470 nm; 1100 µW/cm 2 ).<br />
The chromatic beam splitters redirect light from the mercury lamp to be used<br />
as fluorescence excitation light (wavelengths smaller than 365 nm and 460 nm<br />
respectively). The bandpass filters only let bands around 365 nm and 436 nm<br />
wavelength pass. The barrier filters block reflected excitation light from the environment<br />
and transmit only the fluorescence light from the specimen..<br />
Fluorescence spectra can characterize algal species (through their chloroplast<br />
absorption and emission spectra) and provide valuable information on the composition<br />
and photocycle of the Euglena gracilis photoreceptor. E.g. the embedded<br />
rhodopsin molecules can change their conformational state depending on the wavelength<br />
of absorbed light and thus fluoresce differently.<br />
Images during fluorescence microscopy were taken with a Canon digital camera.<br />
5.2.2 Spectroscopy<br />
Some optical spectroscopy was carried out for educational purposes. Analysis of<br />
different parts of the cell was done, using a Jasco 7850 spectrometer and fluorescence<br />
spectra were recorded using a Perkin Elmer LS 50 B fluorimeter in Pisa.<br />
2 Fixated cells are treated in such a way that enzymatic activity is shut down. This prevents<br />
tissue autodigestion (autolysis), i.e. that unregulated enzymes would otherwise start to<br />
decompose cell organelles and membranes.<br />
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