Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells.

Area of Science:

• Cellular Biology
• Biophysics
• Biochemistry

Background:

• Intracellular calcium (Ca(2+)) is a critical second messenger regulating numerous cellular processes.
• Accurate measurement of intracellular Ca(2+) concentration ([Ca(2+)]i) and its dynamics is essential for understanding cell function and dysfunction.
• Fluorescence microscopy with Ca(2+) indicators is a key technique, but intensity-based measurements can be affected by dye concentration variations.

Purpose of the Study:

• To evaluate the feasibility of the fluorescent Ca(2+) indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM).
• To assess the reliability of 2P-FLIM for quantifying intracellular Ca(2+) in biological preparations.

Main Methods:

• Utilized Oregon Green Bapta-1 (OGB-1) as a fluorescent Ca(2+) indicator.
• Employed two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to measure fluorescence decay times.
• Performed in situ calibration within living cells to analyze Ca(2+)-dependent dye behavior.

Main Results:

• OGB-1 exhibited Ca(2+)-dependent biexponential fluorescence decay in aqueous solution, reflecting Ca(2+)-free and Ca(2+)-bound states.
• In situ calibration in cells allowed differentiation of dye states via global biexponential decay analysis, despite reduced sensitivity.
• Successful quantitative measurements of intracellular Ca(2+) and stimulus-induced changes were achieved in salivary gland cells.

Conclusions:

• OGB-1 is a suitable indicator for 2P-FLIM measurements of intracellular Ca(2+).
• 2P-FLIM offers a robust method for reliable Ca(2+) quantification, independent of dye concentration variations.
• This technique enhances the ability to study cellular physiology and dynamics.