Stimulated Raman scattering reveals metabolic dynamics in living cells

14 Aug 2018

Columbia University platform using heavy water as tracer could aid effective removal of tumors

Several bioimaging techniques are potentially able to image the metabolic process underway in living cells, but many of them have some inherent drawbacks, either in terms of their limited spatial resolution or the need to introduce potentially toxic labeling molecules.

A team led by Wei Min at Columbia University has now developed a promising new alternative, by combining stimulated Raman scattering with the use of relatively innocuous deuterium oxide—or heavy water—as a contrast agent, one which is readily incorporated into proteins and lipids during metabolic activities.

The new method, termed deuterium oxide probing and stimulated Raman scattering (DO-SRS), should offer high sensitivity and subcellular resolution, while also being suitable for in vivo live imaging in mammals. The work was published in Nature Communications.

"We can use this technology to visualize metabolic activities in a wide range of subjects," said Wei Min. "By tracking where and when new proteins, lipids and DNA molecules are made, we can learn more about how animals develop and age, and what goes wrong in the case of injury and disease."

Heavy water is already used to label proteins and lipids as a way to track metabolic changes, but the analysis is then usually carried out with a mass spectrometer, on cells extracted from the body. DO-SRS should make it possible to visualize a continuous picture of the processes involving the heavy water happening inside animal cells in situ.

Raman spectroscopy relies on inelastic scattering of incident photons from a laser to reveal information about the vibrational modes of biological molecules, and hence identify which chemical bonds are present. Stimulated Raman scattering (SRS) employs an additional laser to create non-linear optical effects in the sample, transforming more of the incident light into useful Raman scattering and improving the signal to noise ratio.

Identifying tumor margins

In DO-SRS, enzymatic incorporation of deuterium from D₂0 into macromolecules generates carbon–deuterium (C–D) bonds, which can then be imaged by SRS. In trials, the DO-SRS technique was applied to roundworms, mice, and zebrafish embryos, revealing details of the metabolic operations underway in the animals.

"Within the broad vibrational spectra of C–D bonds, we discovered lipid-, protein-, and DNA-specific Raman shifts, and developed spectral unmixing methods to obtain C–D signals with macromolecular selectivity," commented the team in its published paper. "DO-SRS microscopy enables us to probe lipogenesis, image protein biosynthesis without tissue bias, and simultaneously visualize the different dynamics of lipid and protein metabolism."

One potential, and immediately valuable, application for DO-SRS could be to visualize tumor boundaries and metabolic heterogeneity, as a way to improve the ability of clinicians to completely remove malignant tissues.

Label-free SRS can already assist in identifying a boundary between malignant and healthy tissues in certain scenarios, through a difference in lipid and protein composition, but DO-SRS may reveal more precisely the higher metabolic activities within a tumor, and hence allow its exact margins to be known.

The project team used DO-SRS to observe brain and colon tumors in mice, and found a clear border becoming visible around the tumors, as more deuterium became incorporated into the newly made proteins and lipids of the cancerous cells.

"This method creates a sharp line between healthy and cancerous tissue, making it much easier to remove the tumor," said Columbia's Lingyan Shi.