Quantitative intracellular calcium imaging with laser-scanning confocal microscopy

Area of Science:

• Biophysics
• Cell Biology
• Cardiology

Background:

• Laser-scanning confocal microscopy is crucial for visualizing cellular processes.
• Calcium (Ca2+) signaling is vital in cardiac myocyte function.
• Single-wavelength indicators like Fluo-3 offer advantages but require calibration for quantitative analysis.

Purpose of the Study:

• To develop a protocol for quantitative analysis of Fluo-3 fluorescence in cardiac myocytes.
• To establish a method for accurately measuring intracellular calcium (Ca2+) changes.
• To demonstrate the application of this method in spontaneously active cardiac cells.

Main Methods:

• Co-loading isolated cardiac myocytes with two Ca2+-sensitive fluorescent indicators: Fluo-3 and Fura-2.
• Utilizing wide-field ratiometric measurements of Fura-2 fluorescence to establish a baseline intracellular calcium (Ca2+) concentration.
• Employing laser-scanning confocal microscopy to visualize Fluo-3 fluorescence and correlate it with Fura-2 baseline measurements.

Main Results:

• A reliable protocol was established for deriving quantitative intracellular calcium (Ca2+) information from Fluo-3 fluorescence.
• Changes in Fluo-3 fluorescence were directly expressed as changes in intracellular calcium (Ca2+) concentration.
• The method was successfully applied to visualize Ca2+ changes in spontaneously active cardiac cells.

Conclusions:

• Co-loading with Fura-2 provides a robust method for quantitative analysis of single-wavelength indicators like Fluo-3 in cardiac myocytes.
• This technique enables accurate measurement of dynamic intracellular calcium (Ca2+) changes.
• Fluorescence energy transfer between Fluo-3 and Fura-2 was not a significant issue in co-loaded cardiac cells under the employed conditions.