Thesis-PDF - IAP/TU Wien

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