152 4.7  Tools Using the Inelastic Scattering of Light

then an enhanced Raman spectrum can be generated for that molecule. The enhancement

allows sample volumes of ~10⁻¹¹ L to be probed at concentrations of ~10⁻¹⁴ M, sufficiently

dilute to permit single-​molecule detection (for a review, see Kneipp et al., 2010).

SERS has been applied to detecting nucleotide bases relevant to DNA/​RNA sequencing,

amino acids, and large protein structures such as hemoglobin, in some cases pushing the

sample detection volume down to ~10⁻¹³ L. It has also been used for living cell samples, for

example, to investigate the process of internalization of external particles in eukaryotic cells

of endocytosis (see Chapter 2).

SERS can also be used in conjunction with microscale tips used in AFM (see Chapter 6).

These tips are pyramidal in shape and have a height and base length scale of typically several

microns. However, the radius of curvature is more like ~10 nm, and so if this is coated in gold

or silver, there will be a similar SERS effect, referred to as tip-​enhanced Raman spectroscopy

(TERS), which can be used in combination with AFM imaging. Recently, carbon nanotubes

have also been used as being mechanically strong, electrically conductive extensions to

AFM tips.

SERS has also been performed on 2D arrays of silver holes nanofabricated to have diameters

of a few hundred nanometers. The ability to controllably nanofabricate a 2D pattern of holes

has advantages in increased throughput for the detection of biological particles (e.g., of a

population of cells in a culture, or a solution of biomolecules), which facilitates miniaturiza­

tion and coupling to microfluidics technologies for biosensing application. Also, although still

in its infancy, the technology is compatible with rendering angle-​resolved Raman scattering

signals in using a polarized light source, which offers the potential for monitoring molecular

orientation effects.

4.7.3  RAMAN MICROSCOPY

A Raman microscope can perform Raman spectroscopy across a spatially extended sample

to generate a spatially resolved Raman spectral image. Raman microscopy has been used

to investigate several different, diverse types of cells grown in culture. For example, these

include spores of certain types of bacteria, sperm cells, and cells that produce bone tissue

(osteocytes). In its simplest form, a Raman microscope is a modified confocal microscope

whose scattered light output captured by a high NA objective lens is then routed via an

optical fiber to a Raman spectrometer. Devices typically use standard confocal microscope

scanning methods.

This is an example of hyperspectral imaging or chemical imaging. In the case of Raman

microscopy, it can generate thousands of individual Raman spectra across the whole of the

field of view. The molecular signatures from these data can then, in principle, be extracted

computationally and used to generate a 2D map showing the spatial localization and con­

centration of different biochemical components in cellular samples. In practice, however, it

is challenging to extract the individual signature from a complex mix of anything more than

a handful of different biochemical components due to the overlap between Raman scatter

peaks, and so the method is largely limited to extracting strong signals from a few key bio­

chemical components.

Hyperspectral imaging is a slow technique, limited by the scanning of the sample but

also in the required integration time for a complete Raman spectrum to be generated for

each pixel in the digitized confocal Raman map. A typical scan for a small pixel array can

take several minutes. This increased exposure to incident light increases the risk of sample

photodamage and limits the utility of the technique for monitoring dynamic biological

processes.

Improvements in sampling speed can be made using direct Raman imaging. Here, only a

very narrow range of wavenumbers corresponding to a small Raman scattering bandwidth is

sampled to allow a standard 2D photodetector array system, such as an EMCCD camera, to

be used to circumvent the requirement for mechanical scanning and full Raman spectrum

acquisition, for example, to monitor the spatial localization of just a single biochemical com­

ponent such as cholesterol in a cell. Also, the temporal resolution is improved using related