Biology Reference
In-Depth Information
Depending on the tissue, there are alternative approaches to estimate F
max
;in
some nerve cells, rapid frequent stimulation of cells can generate intracellular Ca
2
þ
levels that approach saturation of the dye (
Maravall et al., 2000
), thus allowing
F
max
values to be estimated.
XVIII. Estimation of
F
min
or the Dynamic Range of the Dye
cult. The ratio of
F
max
/F
min
measured outside the cell cannot be assumed to apply inside; estimates
suggesting that values of
Estimation of F
min
or the dynamic range of the dye is more di
Y
70-80% of those seen in free solutions are common
(
Poenie, 1990
). Again, an averaged value can be obtained using patch pipettes as a
means of establishing bu
ered [Ca
2
þ
] inside cells. Alternatively, the F
min
in each
experiment can be estimated if the intracellular Ca
2
þ
can be lowered to 1-10 nM
(for Fluo-3) by decreasing extracellular Ca
2
þ
, but this assumes that intracellular
Ca
2
þ
can be readily manipulated by changes in the extracellular environment
which is not always the case for every cell type. The e
V
ect of under- or overestima-
tion of the dynamic range of the indicator is shown in
Fig. 10
C. Based on the
reported dynamic range of Fluo-based dyes (
V
), large under/overestimates of
the dynamic range (by up to 30%) cause only small errors in Ca
2
þ
estimation and
only in the lower range of [Ca
2
þ
] values relative to the K
d
of the dye.
100
XIX. Consequence of Errors in Estimation of Intrinsic and
Dye Fluorescence
Prior to conversion of the indicator-based fluorescence to [Ca
2
þ
], the back-
ground or intrinsic fluorescence of the cell/tissue has to be subtracted from the
signal. All cells have an intrinsic fluorescence mainly due to the metabolites beta
nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide
(FAD); their excitation wavelengths are 350-500 nm and emission wavelengths
450-600 nm. The relative fluorescence of these two metabolites depends on the
metabolic state of the cell/tissue and degree of photobleaching. Thus, intrinsic
cellular fluorescence is significant and variable. The most advisable approach is to
use a dye with a significant basal fluorescence that is many times (
) that of the
intrinsic value. This cannot always be achieved; the Fluo-based and Rhodamine-
based dyes are by far the most popular dye groups used in confocal and 2P excitation
microscopy. Their main attraction is a large dynamic range as a result of a low
fluorescence signal from the Ca
2
þ
free form. In this situation, F
min
values are fre-
quently comparable to that of the intrinsic fluorescence of the cell and therefore it is
important to quantify either by parallel measurements on nonloaded tissue or from a
single cell prior to the introduction of the dye. Error in estimation of background
fluorescence (which can be up to 100%) has dramatic e
>
10
V
ects on the calculation of
