Close

Search

Search Menu
Photonics Media Photonics Buyers' Guide Photonics EDU Photonics Spectra BioPhotonics EuroPhotonics Industrial Photonics Photonics Showcase Photonics ProdSpec Photonics Handbook
More News
share
Email Facebook Twitter Google+ LinkedIn Comments

  • Modified GFP enables in vivo imaging of cellular signaling in the heart

BioPhotonics
May 2006
Gwynne D. Koch

The efficient functioning of the heart — an organ that begins to operate before it is fully developed — depends on the coordinated release and reuptake of calcium ions from organelles within cells. Subtle dysfunctions of this process can result in irregular heartbeat and even death.

Fluorescence imaging using indicator molecules that bind to calcium ions has provided some insight into the regulatory processes underlying signaling events in single cells. However, these approaches have limitations when applied to complex, multicellular organs such as the beating heart.

According to Michael I. Kotlikoff of Cornell University in Ithaca, N.Y., there also is no way to introduce a dye into a working heart in vivo. Although the heart can be removed from the body and perfused with a dye, allowing a brief period of signal recording, the loading of the dye is often uneven and the fluorescent signal cannot be isolated to target cells.

Kotlikoff and colleagues at several other universities have created an improved calcium-ion sensor by modifying GFP. The sensor molecule exhibits greater brightness and stability, enabling examination of signaling events in heart cells in vivo. They genetically engineered mice to express the fluorescent molecule when calcium concentrations in heart cells increase, which occurs when the muscle contracts. The advantage of genetically encoding the sensor is that the molecule is always present at the same concentration in cells and is confined to the cells of interest.


GFP was modified to create a brighter and more stable calcium-ion sensor that enables recording of cellular signaling in mouse hearts in vivo. The sequential images show calcium-ion-dependent fluorescence of the sensor molecule during a single cardiac cycle.

Calcium ions signal the heart to contract and determine the force of the contraction. Recording the intensity of the sensor molecule’s fluorescence enabled the researchers to monitor the pattern, rate and force of the contractions. The molecule was excited at 488 nm with a mercury lamp, and emission was collected above 500 nm using a cutoff filter on a wide-field dissecting microscope from Leica Microsystems of Wetzlar, Germany.

Because mouse hearts beat rapidly — about six to 10 times per second — images were captured at 66 and 128 fps using a high-speed electron-multiplying CCD camera — cooled to —90 °C — from Andor Technology of Belfast, UK.

Motion is a persistent difficulty when imaging in vivo. To limit motion, the researchers packed the chest with gauze to stabilize the heart and occasionally increased the anesthesia, which slowed the heart to a more reasonable rate of five to six beats per second. They also imaged at 128 or 256 Hz.

The study demonstrates that a genetically encoded sensor molecule can be used to monitor cellular calcium-ion signaling events in vivo, such as those that occur during development and disease processes, in complex, multicellular organs. The team is experimenting with transplanting embryonic cells from the mice into damaged tissues to determine the effectiveness of cell-based therapies and to see what happens to the cells electrically after transplantation. They also are engineering mice to express the sensor molecule in neurons and in smooth-muscle, conducting and endothelial cells.

PNAS, March 21, 2006, pp. 4753-4758.


Comments
Terms & Conditions Privacy Policy About Us Contact Us
back to top

Facebook Twitter Instagram LinkedIn YouTube RSS
©2016 Photonics Media
x Subscribe to BioPhotonics magazine - FREE!