Improvements to Raman spectroscopy using laser frequency combs allow multiple signals from different parts of a molecule — or even different molecules — be monitored simultaneously using a single detector. The advance is seen as a major step toward the holy grail of real-time, label-free biomolecular imaging.
For decades, biologists have attached fluorescent dye labels to certain proteins to distinguish them under a microscope. But such labels can alter the cell’s function. Many molecules of interest have characteristic absorption spectra at mid-infrared wavelengths, but such long wavelengths do not allow for good spatial resolution.
Probing the heart beat of molecules in a liquid sample. Courtesy of MPQ.
For label-free imaging, biologists have long used coherent Raman spectroscopy, which obtains chemical and structural information about molecules by analyzing how laser light is scattered by a sample.
Because the technique uses near-infrared or visible lasers, it offers high spatial resolution and three-dimensional sectioning capability. But scanning Raman microscopes focus mostly on a distinct spectral feature of a selected molecular species so that they can provide images quite rapidly. To analyze a complex mixture of molecules with possible unknown components, a complete Raman spectrum must be recorded for each image pixel, something existing techniques are too slow to do.
That’s where the frequency comb comes in. A team at the Laser Spectroscopy Div. of the Max Planck Institute of Quantum Optics (MPQ), led by professor Theodor W. Hänsch and Dr. Nathalie Picqué, showed that these combs, which are lasers that produce a train of ultrashort pulses at a highly precise rate, can speed up impulsive Raman scattering to enable measurement on the microsecond timescale, which is fast enough to be used for microscopy.
Previous work to adapt Raman scattering for microscopy suffered from long time delays and enabled only single components of systems to be imaged at video rates, while the MPQ work can rapidly identify different molecules.
The ability to use microscopy in this way could help explain how a drug influences a living cell, or how signal molecules alter cell metabolism.
The technique uses only a single photodetector to measure a complete spectrum.
“Replacing the detector by a camera would make real-time hyperspectral imaging possible, as we could simultaneously measure as many spectra as there are pixels on the camera,” said Takuro Ideguchi, a doctoral student in the group.
The MPQ method is currently limited by long wait times between successive spectral acquisitions, but the scientists believe this hurdle can be overcome with further development of the system. They expect the technique to offer new possibilities not only in spectroscopy but also in real-time microscopy observations of, for example, biological processes.
“It is exciting that a tool like the laser frequency comb, initially developed for frequency metrology, is finding interdisciplinary applications far beyond its original purpose,” said doctoral student Simon Holzner.
Collaborators on the project, which appears in the Oct. 17 issue of Nature (doi: 10.1038/nature12607), include Ludwig Maximilian University in Munich and the Institut des Sciences Moléculaires d’Orsay in France.
For more information, visit: www.mpq.mpg.de/cms/mpq/en/index.html
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