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 [Reference Saar, Freudinger, Reichman, Stanley, Holtom and Xie1]. 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.
SRS microscopy relies on back-scattering of the forward-directed signal by the tissue sample. In biological tissues the backscattered signal is too weak to be detected by an objective lens to allow for high-speed imaging. This limitation was overcome by placing a photodetector directly in front of the objective lens and sending the excitation probes through an aperture in the center of the detector. This and other modifications allowed greater collection of backscattered signal to increase the imaging speed by three orders of magnitude!
In a proof-of-concept experiment, Saar et al. studied the penetration of a small-molecule drug (trans-retinol, which stimulates collagen synthesis) and showed that in vivo it penetrated mouse skin via the hair shaft, one of three hypothesized penetration routes. Similar studies showed the value of SRS microscopy on human skin in a living volunteer (Figure 1). This powerful label-free imaging modality can now be applied to a broad range of problems in whole living organisms. This is likely to play an increasingly important role in clinical diagnostic procedures.
Figure 1: SRS skin imaging in living humans. (A-C) SRS images of the stratum corneum and viable epidermis tuned into CH3 stretching vibration of proteins (2950 cm−1) showing nuclei of variable size (A and B) as well as a hair (C). (D) SRS image of DMSO penetrating the skin at the same region as (C). DMSO accumulates in the hair shaft. Deuterium labeling was used to create a unique vibration of d6-DMSO at 2120 cm−1 for specific imaging. Images are acquired in the epi-direction on the forearm of a volunteer (the principal investigator). Image acquisition time was 150 ms for (A) and (B) and 37 ms for (C) and (D), all with 512 × 512 pixel sampling. Scale: 50 microns [Reference Saar, Freudinger, Reichman, Stanley, Holtom and Xie1].
