TY - JOUR
T1 - Determining calcium concentration in heterogeneous model systems using multiple indicators
AU - Hyrc, Krzysztof L.
AU - Rzeszotnik, Ziemowit
AU - Kennedy, Bryan R.
AU - Goldberg, Mark P.
N1 - Funding Information:
The authors are grateful to Sally McIver, Jennifer Ness and Mario Valentino for help in preparing the manuscript. This work was supported by grants P01 NS032636 and R01 NS36265 from the National Institutes of Health (to MPG).
PY - 2007/12
Y1 - 2007/12
N2 - Intracellular free calcium concentrations ([Ca2+]i) are assessed by measuring indicator fluorescence in entire cells or subcellular regions using fluorescence microscopy. [Ca2+]i is calculated using equations which link fluorescence intensities (or intensity ratios) to calcium concentrations [G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440-3450]. However, if calcium ions are heterogeneously distributed within a region of interest, then the observed average fluorescence intensity may not reflect average [Ca2+]i. We assessed potential calcium determination errors in mathematical and experimental models consisting of 'low' and 'high' calcium compartments, using indicators with different affinity for calcium. [Ca2+] calculated using average fluorescence intensity was lower than the actual mean concentrations. Low affinity indicators reported higher (more accurate) values than their high affinity counterparts. To estimate compartment dimensions and respective [Ca2+], we extended the standard approach by using different indicator responses to the same [Ca2+]. While two indicators were sufficient to provide a partial characterization of two-compartment model systems, the use of three or more indicators offered full description of the model provided compartmental [Ca2+] were within the indicator sensitivity ranges. These results show that uneven calcium distribution causes underestimation of actual [Ca2+], and offers novel approaches to estimating calcium heterogeneity.
AB - Intracellular free calcium concentrations ([Ca2+]i) are assessed by measuring indicator fluorescence in entire cells or subcellular regions using fluorescence microscopy. [Ca2+]i is calculated using equations which link fluorescence intensities (or intensity ratios) to calcium concentrations [G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440-3450]. However, if calcium ions are heterogeneously distributed within a region of interest, then the observed average fluorescence intensity may not reflect average [Ca2+]i. We assessed potential calcium determination errors in mathematical and experimental models consisting of 'low' and 'high' calcium compartments, using indicators with different affinity for calcium. [Ca2+] calculated using average fluorescence intensity was lower than the actual mean concentrations. Low affinity indicators reported higher (more accurate) values than their high affinity counterparts. To estimate compartment dimensions and respective [Ca2+], we extended the standard approach by using different indicator responses to the same [Ca2+]. While two indicators were sufficient to provide a partial characterization of two-compartment model systems, the use of three or more indicators offered full description of the model provided compartmental [Ca2+] were within the indicator sensitivity ranges. These results show that uneven calcium distribution causes underestimation of actual [Ca2+], and offers novel approaches to estimating calcium heterogeneity.
KW - Calcium domains
KW - Fluorescent indicators
KW - Indicator affinity
KW - Intracellular free calcium concentration measurements
KW - Microscope resolution
UR - http://www.scopus.com/inward/record.url?scp=34748902883&partnerID=8YFLogxK
U2 - 10.1016/j.ceca.2007.02.002
DO - 10.1016/j.ceca.2007.02.002
M3 - Article
C2 - 17376527
AN - SCOPUS:34748902883
SN - 0143-4160
VL - 42
SP - 576
EP - 589
JO - Cell Calcium
JF - Cell Calcium
IS - 6
ER -