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dc.contributor.authorZou, Hui
dc.contributor.authorLifshitz, Lawrence M.
dc.contributor.authorTuft, Richard A.
dc.contributor.authorFogarty, Kevin E.
dc.contributor.authorSinger, Joshua J.
dc.date2022-08-11T08:09:30.000
dc.date.accessioned2022-08-23T16:33:47Z
dc.date.available2022-08-23T16:33:47Z
dc.date.issued2004-09-01
dc.date.submitted2008-10-31
dc.identifier.citationJ Gen Physiol. 2004 Sep;124(3):259-72. <a href="http://dx.doi.org/10.1085/jgp.200409066">Link to article on publisher's site</a>
dc.identifier.issn0022-1295 (Print)
dc.identifier.doi10.1085/jgp.200409066
dc.identifier.pmid15337821
dc.identifier.urihttp://hdl.handle.net/20.500.14038/38181
dc.description.abstractThe feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-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 (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) 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 DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) 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 Ca(2+) 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 Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=15337821&dopt=Abstract">Link to Article in PubMed</a>
dc.subjectAniline Compounds
dc.subjectAnimals
dc.subjectBufo marinus
dc.subjectCaffeine
dc.subjectCalcium
dc.subjectCalcium Channels
dc.subjectElectric Conductivity
dc.subject*Fluorescence
dc.subjectFluorescent Dyes
dc.subjectIon Channel Gating
dc.subjectMicroscopy, Fluorescence
dc.subjectMyocytes, Smooth Muscle
dc.subjectPatch-Clamp Techniques
dc.subjectXanthenes
dc.subjectPhysiology
dc.titleUsing total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events
dc.typeJournal Article
dc.source.journaltitleThe Journal of general physiology
dc.source.volume124
dc.source.issue3
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=2061&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/1062
dc.identifier.contextkey659251
refterms.dateFOA2022-08-23T16:33:47Z
html.description.abstract<p>The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-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 (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) 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 DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) 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 Ca(2+) 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 Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.</p>
dc.identifier.submissionpathoapubs/1062
dc.contributor.departmentDept. of Physiology
dc.source.pages259-72


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