Laser induced calcium oscillations in fluorescent calcium imaging

Area of Science:

• Cell Biology
• Microscopy Techniques
• Calcium Signaling

Background:

• Phototoxicity is a common issue in live cell imaging due to excessive laser exposure.
• This can lead to artifacts, including significant increases in intracellular calcium (Ca2+) concentration, complicating data interpretation.
• Limited information exists on laser- and dye-induced changes in cytoplasmic calcium concentration.

Purpose of the Study:

• To describe laser-induced intracellular calcium fluctuations in live cells.
• To help investigators using laser scanning confocal microscopy distinguish artifacts from real signals.
• To evaluate the impact of low-intensity laser excitation on Fluo-3/4/8 loaded cells.

Main Methods:

• Utilized mouse pancreatic acinar cells loaded with Fluo-3/4/8.
• Applied low-intensity 488 nm laser excitation (ca. 10 µW incident power) at 1 Hz.
• Evaluated fluorescent fluctuations and employed BAPTA-AM treatment and a reactive oxygen species (ROS) scavenger (DMPO).

Main Results:

• Low laser power induced repetitive, 2-3 fold elevations in intracellular fluorescence.
• Fluorescence fluctuations were laser power dependent and linked to Ca2+ release from intracellular stores.
• These artifactual Ca2+ waves were indistinguishable from those evoked by secretagogue stimulation.

Conclusions:

• Laser scanning confocal microscopy, even at low power, can induce Ca2+ artifacts that mimic biological responses.
• Strategies are proposed to differentiate these laser-induced artifacts from genuine Ca2+ signaling.
• Awareness of phototoxicity is crucial for accurate live cell imaging data interpretation.