Laser Technique Monitors Calcium

Richard Gaughan

Changes in cellular calcium concentration affect physiological activities such as muscular contraction and neural transmission, but researchers have been unable to resolve the mechanisms of transmission using chemical, electrical and mechanical stimulation. Now a group in Japan has developed a laser-based technique that generates waves of calcium ions in a cell, enabling better resolution and greater control over the phenomenon in experiments.

Researchers are using a laser-based technique to induce calcium waves in cells. A fluorescence indicator enables them to monitor the spread of the wave. Here the frames are separated by 0.4 s.

The setup incorporates a Spectra-Physics Tsunami Ti:sapphire laser and an Olympus Optical fluorescence microscope, focusing the 80-fs pulses of 780-nm light through the 60X, 0.9-NA objective lens of the microscope. Although the near-infrared radiation has a low absorption cross section within the cell, the intensity is high enough to induce two-photon absorption in a 700-nm region near the focus and thus generates calcium waves.

In an initial study, the researchers from Osaka University and from Kyoto Prefectural University of Medicine in Kyoto used cancerous epithelial cells that they had infused with a fluorescent Ca²⁺ indicator. They varied the laser exposure time on the cells from 0.008 to 0.5 s.

A mercury lamp induced fluorescence in the indicator as it spread through the cells, which an intensified CCD camera from Hamamatsu Photonics KK imaged.

With an exposure time of 0.5 s, the laser delivered 37 mW in 42 million pulses to a location 2 µm inside the cell membrane, inducing a calcium wave to spread through the cells. Additional tests demonstrated that calcium waves could be generated when the laser illuminated the interface between adjacent cells.

The researchers expect that the photogeneration of calcium waves will be a valuable tool for investigating the importance of calcium concentration within and among cells. They reported their findings in the Aug. 20 issue of Applied Physics Letters.