1. Goals
Bacteria, marine plankton but also 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 is such cells is to determine autofluorescence above 600nm simultaneously with the fluorescences of functional or other biochemical stains below 600nm (Tab.2).
2. Practical Concepts
In case of UV-excitation (350-370nm), fluorescence emission from
added functioanl dyes like:
INDO1 for Ca2+
ADB for intracellular pH and
OPT for intracellular glutathione and free protein SH-groups
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 FITC-excitation (488nm), functional dye fluorescence of
e.g:
DiOC6(3) for transmembrane potential
R123 mitochondrial potential
DHR sensitive H2O2/peroxidase activity
DCFH-DA H2O2/peroxidase
HE O2-general oxidation
R110 substrates cysteine + serine proteinases
EB DNA dead cells
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.