Stimulated Raman scattering microscopy with long wavelengths for improved imaging depth

Stimulated Raman scattering (SRS) imaging is a fast, label-free, and sensitive technique to map the distribution of a vibrational species in a microscopy setting. It has great potential for applications in many fields, such as lipid imaging in biomedicine. However, depth penetration of the light into the sample is an issue with any light-based technique, especially with multiphoton techniques such as SRS. Using longer wavelengths allows deeper penetration into densely scattering materials, but applying wavelengths above 1,500 nm is challenging technically. We have built a flexible SRS microscope system capable of imaging with a combination of 1,064 nm and wavelengths over 1,500 nm, using the idler output of an optical parametric oscillator (OPO). For comparison, the same system was also operated in the more common configuration, using 1,064 nm in combination with the OPO signal output around 800 nm. With the long-wavelength settings, we show improved depth penetration in polyethylene plastic material and in a silicone phantom with embedded polymer microbeads, and we report images of lipid structure in biological tissue. These results demonstrate the technical feasibility of using these long wavelengths for SRS imaging. Disadvantages such as poorer spatial resolution and lower signal strength are also discussed. The application of this new approach to SRS microscopy can allow greater insight into deep-lying structures in a non-invasive way.