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Address correspondence to Hui Zou, Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Fax: 508-856-5997; email: imaging.ionchannels{at}umassmed.edu
The feasibility of determining localized Ca2+ influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca2+ fluorescence transients (SCCaFTs), due to Ca2+ entry through single openings of Ca2+-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (
Ftotal or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca2+ influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that Ftotal is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca2+ influx and Ftotal was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca2+ was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca2+ current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca2+ current underlying Ca2+ sparks resulting from Ca2+ release from intracellular stores via ryanodine receptors in the same preparation.
Key Words: single channels • calcium imaging • SCCaFT • buffer binding capacity • smooth muscle
1 To simplify the notation, all of the variables with a "
" prefix are time dependent except when denoted with "max" in the subscript.
2 The construction of fluorescence images from either confocal or wide-field microscopy usually involves normalizing the fluorescence at each pixel to the resting fluorescence as F/F0 or F/F0. For wide-field fluorescence imaging of a macro fluorescence event occurring throughout the cell as would occur by activation of whole-cell Ca2+ current in a spherical or cylindrical cell, this procedure is appropriate in order to normalize for the effect of indicator concentration and/or cell depth. It might also be appropriate for confocal imaging of localized fluorescence events to normalize for the effect of indicator concentration where the fluorescence from each pixel is theoretically from a thin slice of the cell. However, the normalization of localized fluorescence events obtained with wide-field imaging can be misleading. This occurs because the same event (F) occurring near the center of the spherical or cylindrical cell (the thicker part of the cell) would produce a smaller percent increase in fluorescence than at the edge (the thinner part of the cell) (Zou et al., 1999). Therefore, we chose to display most of our images without normalization by the resting fluorescence and used only the increase in fluorescence, F, instead.
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