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Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events
Hui Zou1, Lawrence M Lifshitz, Richard A Tuft
1Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655, USA. imaging.ionchannels@umassmed.edu
The Journal of General Physiology
|September 1, 2004
Summary
This study demonstrates a new method to measure calcium (Ca2+) influx using wide-field fluorescence imaging. This technique quantifies localized calcium entry through ion channels, offering a simpler approach than confocal microscopy.
Area of Science:
- Cellular physiology
- Biophysics
- Fluorescence imaging
Background:
- Quantifying localized calcium (Ca2+) influx is crucial for understanding cellular signaling.
- Existing methods often require complex imaging techniques like confocal microscopy.
- Wide-field fluorescence imaging offers potential for simpler, more accessible measurements.
Purpose of the Study:
- To explore the feasibility of determining localized Ca2+ influx using only wide-field fluorescence images.
- To develop a method for measuring single channel Ca2+ fluorescence transients (SCCaFTs).
- To establish a relationship between Ca2+ influx and measurable fluorescence signals.
Main Methods:
- Imaging of SCCaFTs using fluo-3 dye.
- Recording of unitary channel currents.
- Utilizing wide-field optics to measure total fluorescence increase (DeltaF(total)) as "signal mass" by summing pixel fluorescence.
- Employing 2D and 3D imaging to assess the independence of DeltaF(total) from channel position.
- Establishing a linear relationship between Ca2+ influx and DeltaF(total) using specific ion channels.
Main Results:
- Demonstrated that DeltaF(total) is largely independent of the channel's position relative to the focal plane.
- Established a linear relationship between Ca2+ influx and DeltaF(total) for caffeine-activated channels.
- Identified factors influencing the "converting factor" (slope) including imaging setup, experimental conditions, and indicator properties.
- Successfully estimated Ca2+ current and endogenous buffer binding capacity.
Conclusions:
- Wide-field fluorescence imaging provides a feasible method for quantifying localized Ca2+ influx.
- The "signal mass" (DeltaF(total)) approach simplifies measurements compared to confocal microscopy.
- This method allows for accurate estimation of Ca2+ currents and buffer properties, including those underlying Ca2+ sparks.