Chemical analysis in vivo and in vitro by Raman spectroscopy--from single cells to humans

Abstract

The gold standard for clinical diagnostics of tissues is immunofluorescence staining. Toxicity of many fluorescent dyes precludes their application in vivo. Raman spectroscopy, a chemically specific, label-free diagnostic technique, is rapidly gaining acceptance as a powerful alternative. It has the ability to probe the chemical composition of biological materials in a non-destructive and mostly non-perturbing manner. We review the most recent developments in Raman spectroscopy in the life sciences, detailing advances in technology that have improved the ability to screen for diseases. Its role in the monitoring of biological function and mapping the cellular chemical microenvironment will be discussed. Applications including endoscopy, surface-enhanced Raman scattering (SERS), and coherent Raman scattering (CRS) will be reviewed.

Figure 1

Schematics of different Raman scattering processes discussed in this article. The excitation laser beam is isotropically scattered by the vibrations of chemical bonds in spontaneous Raman scattering (top). The same objective lens can used for the excitation and collection of the Raman signal, with a dichroic mirror serving as a means of separating the two beams. When two or more coherent laser beams with different frequencies overlap spatially and temporally onto the specimen, a coherent Raman signal on the anti-Stokes side of the spectrum is generated (middle). This CARS signal is produced by nonlinear mixing of the ultrashort excitation pulses and is several orders of magnitude stronger than the spontaneous Raman signal. Molecules adsorbed on metallic nanostructures will experience a strong electromagnetic field in addition to chemical enhancement, due to the presence of plasmon resonances near the surface of metals (bottom). The Raman signal can be enhanced by up to 15 orders of magnitude in this way.

Figure 2

Diagram representing a normal (top) and diseased (bottom) cell in culture with their corresponding Raman spectra. The spectra were obtained with a 785nm continuous-wave laser beam that simultaneously optically traps the cells and generates the Raman signal. Typical protein peaks can be observed around 1442 cm⁻¹ and 1001 cm⁻¹ and DNA peaks around 785 cm⁻¹, 1090 cm⁻¹, and 1575 cm⁻¹. Significant differences between the normal and cancerous cell can be observed in the Raman spectrum for the peaks centered on 1575 cm⁻¹, and 1090 cm⁻¹, showing the capability of Raman spectroscopy to distinguish the two types of cells.

Figure 3

Schematic of a Raman endoscope recording spectra from the lung (top) and bone (bottom). The spectra shown in this figure were obtained by a fiber-based Raman probe using a 785 nm continuous-wave excitation laser and a standard holographic grating spectrometer. The lung and bone specimens were excised mouse tissue samples. The lung spectrum shows significant similarities with the spectra of single cultured cells, reflecting the protein and DNA content of the sample. The bone spectrum, on the other hand, contains Raman peaks specific for calcium phosphate groups.