Raman spectroscopy and optical trapping combined to study mitochondria

Gary Boas

Every year, almost 4000 children in the US are born with a mitochondrial disease, and in developing countries, the illnesses are even more common. Mitochondria play an important role in energy metabolism during cell life as well as during cell death, and damage or dysfunction in mitochondria can contribute to diseases such as cancer and diabetes as well as to cardiovascular and age-related neurodegenerative diseases. For this reason, researchers have devoted considerable effort to studying the structure and function of single mitochondria. Improved understanding in these areas could aid in the development of drugs to treat these diseases.

Previous investigations have used Raman spectroscopy to explore the molecular composition and conformation that determine the function and bioactivity of mitochondria. But recently a team with the Chinese Academy of Sciences in Beijing, with the Guangxi Academy of Sciences in China, and with East Carolina University in Greenville, N.C., used Raman spectroscopy and an optical trap to study individual, isolated mitochondria in an experiment performed at the biophysics laboratory of the Guangxi Academy of Sciences.

Investigators have combined Raman spectroscopy and optical trapping to facilitate the study of individual, isolated mitochondria after adding a calcium-ion solution. Shown here are Raman spectra of single trapped mitochondria, both intact (at 0 min) and at various times after adding the solution, which induces swelling. Reprinted with permission of Optics Express.

Many previous studies of mitochondrial composition and bioactivity have been based on ensemble measurements, where researchers cannot determine precisely the contributions of individual mitochondria. “This is a particular problem with activation and inactivation of mitochondria, since the kinetics of these processes can be heterogeneous between individual mitochondria in a population,” said Yong-qing Li, a member of the physics department at the university and one of the authors of the study. Combining Raman spectroscopy and optical trapping enabled the investigators to observe properties of individual mitochondria that would have been masked in ensemble measurements.

To demonstrate the method — which they call laser tweezers Raman spectroscopy — the researchers used it to trap a single mitochondrion and to measure its Raman spectra after adding a calcium-ion solution. The system they used consisted of a Sacher Lasertechnik LLC semiconductor diode laser beam operating at 785 nm introduced into a Nikon inverted differential interference contrast microscope with a 100×, 1.30-NA objective to create an optical trap. The same beam excited Raman scattering of the trapped mitochondrion, and the objective collected the scattering light. A liquid nitrogen-cooled CCD made by Princeton Instruments of Trenton, N.J., obtained the spectra.

The investigators demonstrated that they could capture single mitochondria for long-term observation, which provided optimum excitation and collection for Raman scattering in a confocal configuration. “Without optical trapping, the small mitochondrial particles (with a size ranging from 0.5 to 5 μm in diameter) would drift away from the excitation beam in the buffer solution,” Li said. Also, the near-infrared light used introduced a minimal amount of damage to the trapped mitochondria.

Having validated the technique, the researchers are planning two separate applications. First, they want to analyze mitochondria from cancer, diabetes and various other diseases to determine the vibrational spectroscopic features of the damaged and defective mitochondria. Also, they hope to use the technique to study mitochondrial drug-targeting strategies that would enable them to manipulate mitochondrial functions to permit the selective protection or eradication of cells, thus advancing therapies for a variety of diseases.

Optics Express, Oct. 1, 2007, pp. 12708-12716.