Molecules Made Mini Lasers

A new optical microscopy technique squeezes photons out of nonfluorescent molecules to provide 3-D images of living cells and tissues for applications in medical imaging and biological research.

The method, developed by Xiaoliang Sunney Xie and colleagues including Wei Min at Harvard University, exploits stimulated emission, during which a photon hitting an atom causes an electron to drop an energy level and results in the creation of another photon. Stimulated emission can be applied to all molecules, which gives the technique a big advantage over other microscopy methods that either use fluorescent molecular tags (fluorophores) or use nonfluorescent techniques that are much less sensitive.

Ex vivo stimulated emission image of blood vessels of a mouse ear based on natural hemoglobin contrast (in red color), surrounding sebaceous glands (green overlay based on confocal reflectance) (Images: W. Min/S. Lu, Harvard University)

Using stimulated emission could give researchers the opportunity to study many nonfluorescent colorful molecules that have not, until now, been seen in superresolution microscopy.

In a paper appearing in the Oct. 22 issue of Nature, Sunney Xie and his group irradiated molecules in a biological specimen with ultrashort pulses of light. Molecules that absorb a photon enter an excited state, and are then irradiated by another light pulse of slightly lower energy. When a photon from the second pulse interacts with an excited molecule, the molecule relaxes back into its ground state by emitting a duplicate photon. The excitation pulse photon is absorbed and lost, while the second-pulse photon adds a twin to the transmitted light, and the gain in photons is measured to quantify the number of molecules in the sample.

Ex vivo stimulated emission image of blood vessels of a mouse ear based on natural hemoglobin contrast (in red color), showing individual blood cells in the vessel network (inset) surrounding sebaceous glands (green overlay based on confocal reflectance)

The researchers show that the technique can be used to monitor the delivery of a particular nonfluorescent drug across the skin, highlighting distribution of the blue dye at both cellular and tissue levels. They also demonstrate detailed imaging of the blood vessels in a nude mouse ear, tracking the position of single red blood cells within individual capillaries and raising the prospect of 3-D mapping of blood oxygenation levels.

"...Min and colleagues’ method is a bold step towards unveiling details of live cells and tissues that would otherwise be left uncharted," said Stefan W. Hell and Eva Rittweger in a "News and Views" article appearing in the same issue.

"An intriguing possibility for the future would be to design a set of laser pulses that fulfill both roles of stimulated emission — switching off molecular signals and stimulating photon emission — to provide images of unlabelled, nonfluorescent molecules at subdiffraction (nanoscale) resolution for the first time," added Hell and Rittweger, who work in the department of nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Earlier this year in another Nature paper, Sunney Xie and colleagues described a microscopy technique based on stimulated Raman scattering that achieves label-free imaging with very high sensitivity.

For more information, visit: www.harvard.edu or www.nature.com