Page 182 - Vibrational Spectroscopic Imaging for Biomedical Applications

158 Cha pte r S i x

6.2 Generation of Raman Images
Raman images have evolved over many years, and can be generated
using several different modes. The modes in which Raman dispersive
spectra are acquired from several adjacent locations within a sample and
then stitched together to form an image are called mapping experiments.
Employment of specialized optical components in order to collect an
image without moving the sample is often called widefield imaging.

6.2.1 Point Mapping
In point mapping, a Raman image is acquired one pixel at a time by
using a point-focused laser beam to collect Raman spectra of adjacent
pixels in a grid pattern. This is still the most commonly used mode of
generating a Raman image, and has been employed to collect images
1–4
of a variety of materials, including human cells, yeast and plant
7
5
5
5,6
cells, biopsy tissues, food materials, tissue adhesives, emulsions
and resins, and carbon nanotubes. Point mapping has been used
8,9
10
in conjunction with other techniques such as surface enhanced Raman
scattering (SERS) 5,11,12 and tip enhanced Raman experiments. 13,14
The point-mapping approach suffers from several technical
drawbacks. Up until now, acquisition of a full complement of spectra
in confocal mode to construct an image has taken a long time. The
time of acquisition cannot be reduced by increasing laser power
because the high laser power density in confocal spectroscopy may
damage samples. As a result, generated images have tended to have
low-image definition. Furthermore, because of the large time of
acquisition, it is commonly suspected that the first few pixels of data
acquired may have differing background influences than those at the
end of the sample grid. Recent improvements in Raman spectrometers
and decreasing time of spectral acquisition, will no doubt have an
impact on images generated by point mapping. Other factors which
impact the quality of point-mapping images are the limitation of
spatial resolution of an image by the size of the laser spot and the
microscope stage’s mechanical capabilities.

6.2.2 Line Mapping
In line mapping, some of the point-mapping issues are overcome.
During excitation, the laser beam is spread into a line with the use of
optics, reducing the laser power density and potential for sample
damage. At the same time, because a large number of pixels are being
illuminated at once, acquisition of the full image tends to be faster. In
addition, the optics required to generate line-scanned Raman maps
are commercially available. Line mapping has been typically performed
by scanning a laser spot with a scanning mirror or using cylindrical
optics. 15,16,17 Further specialized optics have been noted to increase beam
uniformity across a line. 18,19 Line mapping has been demonstrated