Coherent Anti-Stokes Raman Scattering
While Raman spectroscopy offers great intrinsic specificity unmatched by any other non-invasive technique, it often lacks the signal strength required by many biomedical applications for fast, real-time measurements. In this case, nonlinear optical approaches come to the rescue. By using tunable short-pulsed lasers, several Raman resonances can be probes simultaneously, leading to amplified signals on the Stokes as well as the anti-Stokes scattered part of the Raman spectrum. We summarize these approaches under the term "Coherent Raman scattering (CRS)".
One approach to CRS is based on a pump-probe spectroscopy technique called coherent anti-Stokes Raman scattering (CARS). In CARS a signal is generated when two lasers are focused into the sample, one acting as the source leading to Stokes-shifted Raman scattering, while the other one is tuned to the same frequency as a specific Raman resonance. The two lasers interact nonlinearly within their overlap volume, simultaneously exciting the characteristic chemical vibration, and driving the amplification of the corresponding anti-Stokes peak (see the sketch in the figure above). The resulting CARS signal is more than a thousand times stronger than the original Raman signal and can be used for chemically selective imaging without the use of fluorescent molecules.
By significantly reducing the exposure time required to record the signal, CARS microscopy holds great promise for imaging living cells and tissues. By keeping the laser powers at a minimum, cells can potentially be imaged chemically and followed over very long durations - hours and days, if needed, because the signal never fades as is the case in fluorescence microscopy.
The CRS microscopy system available in our laboratory is quite unique in that it combines three short-pulsed lasers, two of which are freely tunable in a range from 770 nm - 960 nm and one is fixed at 1064 nm. The combination of these lasers allows us to simultaneously probe two Raman resonances and create ratiometric image contrast, or to utilize other four-wave mixing processes to exploit interference phenomena between different Raman signals. This enables us to achieve the highest possible sensitivity and specificity in imaging biological samples. Also, the detection of the Raman signals can be done with either analog or digital (photon-counting) detectors. This allows us, for example, to separate the instantaneous Raman-scattered signals from background signals due to two-photon excited fluorescence, which are typically delayed by a few nanoseconds. It enables us to collect multiphoton-excited fluorescence and CARS signals from the same sample, e.g. facilitating simultaneous confocal fluorescence and CARS imaging by using temporal gating of the signal photons. This combination allows us to extract the CARS signal even from highly autofluorescent samples, such as arterial thin sections, where the fluorescence and CARS signal overlap spectrally.
As examples of our recent work in this area, we have e.g. shown that CARS, when combined with laser trapping, can assess dynamic chemical alterations in individual lipid particles on the millisecond time scale. We have demonstrated the separation of CARS and autofluorescence signals in highly fluorescent samples, such as plant cells and tissue sections, and we have shown that time-gating can also be used to separate CARS signals with different intrinsic nature, allowing us to image samples with contrast over all length scales.
An example of CARS imaging contrast is shown in the figure to the right. Here, a living human adipocyte (fat cell) derived from a mesenchymal stem cell was imaged by tuning to the 2845 cm-1 lipid vibration as shown in the inset. The CARS process then creates intrinsic contrast for all the lipid droplets that are accumulated in these cells.
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