Microscopy is Only Skin Deep

Abstract

Light optical imaging techniques that don't require labels are attractive for human studies because they are not toxic nor do they perturb the system being studied. Whereas there are several methods available to provide microscopic imaging below the surface of a tissue, they each suffer from limitations, such as low spatiotemporal resolution and a limited number of molecular signatures that can be imaged. Raman spectroscopy offers label-free contrast for major chemical species in tissue, such as water, lipids, DNA, and proteins based on vibrational spectra at light optical wavelengths. However, the Raman signal is very weak, which makes it difficult to image a sample with good temporal resolution. The recent development of stimulated Raman scattering (SRS) microscopy can overcome these limitations as demonstrated by the elegant studies of Brian Saar, Christian Freudiger, Jay Reichman, Michael Stanley, Gary Holton, and Sunney Xie. Saar et al. improved the optics and electronics for the acquisition of the backscattered signal of SRS. In SRS microscopy, the sample is excited by two lasers at different frequencies. When the difference in frequency matches a molecular vibration in the sample, the intensities of the probes change in a predictable manner. These intensity changes are small, but Saar et al. developed a new method to detect them. They chopped one of the laser beams at high frequency (MHz) and detected the intensity change in the other beam, which offered superior sensitivity. The key component was a custom-made all-analog lock-in amplifier with a very short (about 100 ns) response time. The laser probe wavelengths were tuned to match a vibrational frequency of interest and raster-scanned across the sample. Frequencies were tuned to detect CH2 stretching (primarily for lipids), OH stretching (primarily water), and CH3 stretching (primarily proteins) vibrations. Imaging of water is of particular interest in studying the transport of water-soluble compounds such as drugs.