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Raman Scattering Sped Up for Microscopy

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Improvements to Raman spectroscopy using laser frequency combs allow multiple signals from different parts of a molecule — or even different molecules — to 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.

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.

 Probing the heart beat of molecules in a liquid sample.
Probing the heart beat of molecules in a liquid sample. Courtesy of MPQ.


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.

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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 single 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

Published: October 2013
Glossary
metrology
Metrology is the science and practice of measurement. It encompasses the theoretical and practical aspects of measurement, including the development of measurement standards, techniques, and instruments, as well as the application of measurement principles in various fields. The primary objectives of metrology are to ensure accuracy, reliability, and consistency in measurements and to establish traceability to recognized standards. Metrology plays a crucial role in science, industry,...
laser spectroscopy
That part of the science involved in the study of the theory and interpretation of spectra that uses the unique characteristics of the laser as an integral part in the development of information for analysis. Raman spectroscopy and emission spectroscopy are two areas where lasers are used.
photodetector
A photodetector, also known as a photosensor or photodiode, is a device that detects and converts light into an electrical signal. Photodetectors are widely used in various applications, ranging from simple light sensing to more complex tasks such as imaging and communication. Key features and principles of photodetectors include: Light sensing: The primary function of a photodetector is to sense or detect light. When photons (particles of light) strike the active area of the photodetector,...
raman spectroscopy
Raman spectroscopy is a technique used in analytical chemistry and physics to study vibrational, rotational, and other low-frequency modes in a system. Named after the Indian physicist Sir C.V. Raman who discovered the phenomenon in 1928, Raman spectroscopy provides information about molecular vibrations by measuring the inelastic scattering of monochromatic light. Here is a breakdown of the process: Incident light: A monochromatic (single wavelength) light, usually from a laser, is...
spatial resolution
Spatial resolution refers to the level of detail or granularity in an image or a spatial dataset. It is a measure of the smallest discernible or resolvable features in the spatial domain, typically expressed as the distance between two adjacent pixels or data points. In various contexts, spatial resolution can have slightly different meanings: Imaging and remote sensing: In the context of satellite imagery, aerial photography, or other imaging technologies, spatial resolution refers to the...
BiophotonicsmetrologyBioScanEuropelabel-free imaginglaser frequency comblaser spectroscopyLasersMaterials & ChemicalsMax Planck Institute for Quantum OpticsMicroscopymid-infraredmoleculeMPQphotodetectorRaman scatteringRaman spectroscopyResearch & Technologyspatial resolutionspectroscopyTakuro IdeguchiTech PulseTest & MeasurementTheodor Hänsch

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