Biology Reference
In-Depth Information
3. permeabilizing the cell with digitonin to release Fluo-3 to permit estimation
of fluorescence background.
Because the fluorescence intensity measured after Step 3 is just the background
signal (including cellular autofluorescence), whereas the intensity after Step 2 is
(F Mn þ
background), one can obtain F Mn by subtraction. The calibration proce-
dure described here is based on the following assumptions: (1) indicator fluores-
cence intensity is not diminishing rapidly as a result of leakage; (2) the fluorescence
properties (F min , F max , and F Mn ) of the indicator are known from in vitro measure-
ments and are the same in cells as in vitro; and (3) the K d of the indicator is also the
same in cells as in vitro.
Quin2 is an example of an indicator the fluorescence of which is quenched
completely by heavy metal ions. For calibration of such an indicator, see the
review on Quin2 by Tsien and Pozzan (1989) .
B. Calibrating a Ratiometric Fluorescent Indicator
A dual-wavelength ratiometric indicator allows excitation spectral intensity or
emission spectral
erent
wavelengths. If F 1 is the fluorescence intensity at wavelength l 1 , F 2 is the fluores-
cence intensity at wavelength l 2 , and R ¼ F 1 /F 2 , then the free Ca 2 þ concentration
can be shown to be ( Grynkiewicz et al., 1985 )
intensity of the indicator to be monitored at two di
V
Ca 2 þ i ¼ K d R R min
s f ; 2
s b ; 2
½
ð2Þ
R max R
where R min is the limiting value of the ratio R when all the indicator is in the Ca 2 þ -
free form and R max is the limiting value of R when the indicator is saturated with
Ca 2 þ . 22 Experimentally, the factor s f,2 /s b,2 is simply the ratio of the measured
fluorescence intensity when all the indicator is Ca 2 þ -free to the intensity measured
when all the indicator is Ca 2 þ -bound, with both intensity measurements taken at
l 2 . On the right side of Eq. (2) , with the exception of K d , which is an intrinsic
property of the indicator, all other terms are ratios of intensities; in forming these
ratios, problems associated with cell shape and dye concentration changes cancel.
Using Eq. (2) to calculate [Ca 2 þ ] requires that K d be known and that R min , R max ,
and s f,2 /s b,2 be determined experimentally. A typical calibration entails
1. increasing Ca 2 þ permeability of the cell with ionomycin or Br-A23187 in the
presence of ''zero'' extracellular Ca 2 þ (EGTA or BAPTA in nominally Ca 2 þ -
free medium; Section IV.B.2 ), so all intracellular Ca 2 þ could be depleted;
22
Usually, l 1 and l 2 are chosen so that intensity measured at l 1 consists mostly of fluorescence
emitted by the Ca 2 þ -bound form of the indicator, whereas intensity at l 2 consists mostly of fluorescence
from the Ca 2 þ -free form. Choosing the wavelength pair in this way increases the di
V
erence between R min
and R max , making it possible to map [Ca 2 þ ] onto a wider range of R values.
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