Evaluated addition of fluorescein to Synechocystis cells for standardizing 77K fluorescence spectra (excited at 435 nm).
Measured absorption at peak 490nm: 0.53
stock solution was diluted 1:3 -> corrected absorption for stock solution at peak 2.12
from: https://en.wikipedia.org/wiki/Fluorescein
Extinction coefficient at 490 nm = 76900 Mol-1 cm-1
Stock solution concentration:
A=e*L*C
C=A/(e*L)
C=2.12 / (76900 cm-1 M * 1 cm
C=2.76 * 10⁻5 M
C=27.6 uM
For a 1:1 fluorescence(490nm) to chlorophyll a (725 nm) ratio 10uM stock were added to 500 uL Synechocystis (OD 1.0).
Final concentration: 30uM *10 / 500 = 0.6 uM
use 0.5 – 1 uM final concentration for 77K fluorescence spectra.