Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Export

@article{citeulike:695440, abstract = {We describe a method for determining intracellular free calcium concentration ([Ca(2+)]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca(2+)] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca(2+)] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca(2+)] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid). Resting [Ca(2+)] was 32-59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca(2+)] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44-80) and peak [Ca(2+)] changes at zero added buffer (178-312 nM). In young animals (postnatal days 14-17) our results were comparable to previous estimates obtained by ratiometric methods (, Biophys. J. 70:1069-1081), and no significant differences were seen in older animals (P24-28). We expect our method to be widely applicable to measurements of [Ca(2+)] and [Ca(2+)]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.}, address = {Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 USA.}, author = {Maravall, M. and Mainen, Z. F. and Sabatini, B. L. and Svoboda, K.}, citeulike-article-id = {695440}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/10777761}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=10777761}, issn = {0006-3495}, journal = {Biophys J}, keywords = {biophysics, calcium, imaging, indicator, neuroscience, photon, two}, month = {May}, number = {5}, pages = {2655--2667}, posted-at = {2006-06-15 20:54:28}, priority = {0}, title = {Estimating intracellular calcium concentrations and buffering without wavelength ratioing.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/10777761}, volume = {78}, year = {2000} }

TY - JOUR ID - citeulike:695440 L3 - citeulike-article-id:695440 N2 - We describe a method for determining intracellular free calcium concentration ([Ca(2+)]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca(2+)] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca(2+)] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca(2+)] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid). Resting [Ca(2+)] was 32-59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca(2+)] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44-80) and peak [Ca(2+)] changes at zero added buffer (178-312 nM). In young animals (postnatal days 14-17) our results were comparable to previous estimates obtained by ratiometric methods (, Biophys. J. 70:1069-1081), and no significant differences were seen in older animals (P24-28). We expect our method to be widely applicable to measurements of [Ca(2+)] and [Ca(2+)]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging. IS - 5 JF - Biophys J SN - 0006-3495 AD - Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 USA. EP - 2667 TI - Estimating intracellular calcium concentrations and buffering without wavelength ratioing. VL - 78 SP - 2655 KW - biophysics KW - calcium KW - imaging KW - indicator KW - neuroscience KW - photon KW - two AU - Maravall, M AU - Mainen, ZF AU - Sabatini, BL AU - Svoboda, K PY - 2000/05// UR - http://view.ncbi.nlm.nih.gov/pubmed/10777761 ER -