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Microorganisms and Plant Cells
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The biochemical analysis of viable unicellular organisms or plant cells is of increasing interest for environmental but also for biotechnological or medical purposes.
Bacteria, marine plankton or isolated plant cells frequently exhibit a strong natural autofluorescence from chlorophyll or other pigments e.g. phycobiliproteins.
The concept for the flow cytometric determination of functional or constituent parameters of such cells is to determine autofluorescence above 600nm simultaneously with the fluorescences of functional or other biochemical stains below 600nm ( tab.1, tab.2).
In case of UV-excitation (350-370nm), fluorescence emission from added functional dyes like:
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INDO1
for Ca2+ ADB for intracellular pH and OPT for intracellular glutathione and free protein SH-groups DNA |
is collected in fluorescence channels F1 and F2 between 390-440nm and 500-600nm while natural chlorophyll fluorescence is determined in fluorescence channel F3 in addition to forward (FSC) and sideward (SSC) light scatter signals.
In case of blue-excitation (488nm), functional dye fluorescence of e.g:
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FDA
esterase activity in viable cells DiOC6(3) for transmembrane potential R123 mitochondrial potential DHR sensitive H2O2/peroxidase activity DCFH-DA H2O2/peroxidase HE O2-general oxidation R110 proteinase substrates cysteine/serine proteinases, aminopeptidases AO DNA of viable and dead cells as well as PI as DNA counterstain for dead cells in all UV or blue excited cell function assays |
is collected between 510-540nm and 540-580nm in fluorescence channels F1 and F2. Chlorophyll fluorescence is again collected in fluorescence channel F3.
Natural autofluorescence may also occur in fluorescence channels F1 or F2. In this case, the added fluorescent dyes due to their brightness should still give valuable information. The natural fluorescence of each cell cluster in an unstained control sample has to be subtracted from the observed fluorescence of the respective cell cluster in the stained sample.
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