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Researchers in France have developed a nonlinear imaging technique that provides both chemical-composition and molecular-orientation information at subsecond timescales—and that could offer a new window into the progression of some neurodegenerative diseases. [Image: Sophie Brasselet, Institut Fresnel, CNRS, Aix Marseille Université]

Nonlinear-imaging techniques provide sophisticated views of chemical composition, in applications ranging from the industrial to the biomedical. But for some investigations—such as studies of neurodegenerative diseases—the orientation of molecules, in addition to their composition, constitutes a crucial variable. And while polarization-resolved imaging techniques can provide that orientation view, most existing approaches are too slow to capture the rapid molecular dynamics important to a deep understanding of disease.

Scientists in France have now figured out a way around that dilemma (Optica, doi: 10.1364/OPTICA.4.000795). By slipping in a key electro-optic element at the right point, the research team has configured a system that can achieve virtually real-time polarization tuning in several nonlinear-imaging techniques. The result? An ability to track changes in molecular order and orientation on subsecond timescales—while still achieving high-resolution imaging of molecular composition. The team believes that the technique could advance studies of neurodegeneration in lab animals and, perhaps, eventually be used in the clinic, to detect early disease development in humans.

Tuned to resonance

Molecular-imaging techniques that rely on two particular nonlinear optical phenomena, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), work, in different ways, by matching the difference frequency between two synchronized laser beams (a pump beam and a probe or Stokes beam) to the vibrational resonance frequency of the molecule of interest. In both cases, the signal resulting from the beams’ interaction with those molecules is detected as an intensity modulation—at the pump or Stokes beam frequency in the case of SRS, and at the frequency of the nonlinear induced anti-Stokes radiation in the case of CARS.

While the specific measurements are different, both techniques allow label-free, molecule-specific imaging, and have garnered increasing interest, especially in biomedical studies. Further, because the resulting signal is strongly sensitive to the incident light’s polarization depending on the angle of incidence, both approaches open up the prospect of extracting information on molecular orientations in addition to composition.

The problem thus far, though, has been speed. The molecular-order imaging techniques developed to date have generally relied on polarization tuning that takes place on the scale of tens of seconds to minutes. That’s far too slow to capture the subsecond-scale processes important to molecular dynamics in living cells.

The Pockels cell difference

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Sophie Brasselet. [Image: Institut Fresnel, CNRS, Aix Marseille Université]

The French research team, led by Sophie Brasselet of the Institut Fresenel at Aix Marrseille Université, wanted to see if it could achieve faster polarization tuning, and thereby capture information on the orientations of biomolecules as well as their composition. The researchers focused in particular on the SRS technique, in which the signal is measured by the power gain in the Stokes beam at the pump-beam frequency. To enable fast polarization tuning, they added a Pockels cell (an electro-optic modulator) to the pump beam line to act as a polarization modulator at 100-kHz frequencies. A voltage ramp applied to the cell sweeps through the range of linear polarization angles from 0 to 180 degrees, to allow polarization lock-in at specific angles.

After passing through the sample, the polarization-modulated signal is picked up at the end of the line by a photodetector, where the Stokes-beam modulation is read. Because of the polarization lock-in, however, the energy transfer leading to the Stokes-beam modulation happens only where the molecules are aligned. And, because of the rapid modulation frequency of the Pockels cell, both the local order and the mean orientation across a larger area can be probed nearly simultaneously, at subsecond time scales—orders of magnitude faster than in other approaches.

Toward the clinic?

The researchers tested out the system on artificial lipid membranes meant to model the myelin sheathing that covers axons in human neurons. (In some neurodegenerative diseases, such as multiple sclerosis, disease pathology is thought to stem from the progressive disorganization of the lipid layers that make up that sheathing, which ultimately causes the sheath to detach from the axon.) The Brasselet team found that the technique could indeed extract detailed information on deformation and lipid organization in those model multilayer membranes, and even in the single lipid bilayers of red blood cells. Further, while the researchers’ work focused on SRS imaging, they also showed that the same kind of fast polarization tuning was feasible with CARS imaging as well.

In a press release, Brasselet noted that the technique could soon find use in lab studies of myelin-sheath breakdown. For example, living lab mice could be probed through tiny “windows” that expose the brain or spinal cord, a common technique. Looking further ahead, Brasselet believes that the new polarization-resolved Raman imaging could even prove out as a diagnostic tool, if it can be adapted to work with endoscopes or other patient-friendly imaging tools. “Ultimately,” she says, “we would like to develop coherent Raman imaging so that it could be used in the body to detect diseases in their early stages.”