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W idefield Raman Imaging of Cells and T issues 161

Because both spatial dimensions are collected simultaneously in
widefield imaging, the duration of a widefield experiment is proportional
to the number of spectral channels and not to the number of pixels.
This is particularly advantageous when a limited spectral range
provides sufficient chemical and spatial information. By reducing the
number of frames required, experimental time decreases without
losing spatial resolution. The spatial resolution of the widefield
experiment is determined by the combination of diffraction, the CCD
pixel size and the microscope magnification at the CCD. Diffraction
limited resolution has been reported down to 250 nm. 36
Used in conjunction with high efficiency solid state lasers, holo-
graphic notch rejection filters, and CCD detectors, the quality of
widefield Raman images has improved greatly. In addition, the devel-
opment of microscopic imaging has enabled the acquisition of even
higher fidelity information. Widefield Raman imaging has been
38
employed in a variety of applications, including the forensic, phar-
maceutical, 39–44 biomedical 45–47 and threat detection 48,49 industries.

6.3 Raman Imaging of Cells and Tissues
Chemical imaging has its roots in biomedical applications, using
fluorescent labels to map concentrations of calcium ions and other
metabolites. Much of the reported Raman imaging studies on cells
50
and tissues has been carried out by point and line mapping. In con-
trast, the body of work involving widefield Raman imaging for the
analysis of cells and tissues is much less extensive.
Bone is a tissue well suited to Raman techniques. The minerals
in bone are high Raman scatterers that have been found to reflect
the state of the tissue. Otto and coworkers utilized line scanning
51
technology to show the distribution of hydroxyapatite in bone
implants. They were able to ascertain the point in the bone at which
there was a transition from crystalline hydroxyapatite of the
implant coating to the weaker bone tissue material itself. The
Morris group has also employed Raman line mapping to study
bone tissue. In their early work, they reported that observation of
the mineral and protein bands in bone provide information about
20
21
maturity and microstructure of human bone. In further investi-
gations, they utilized line mapping to identify and describe the
effect of fatigue in bone. 22
Krafft and coworkers used point mapping to assess the diagnostic
potential of Raman images to distinguish between normal brain tissue
7
and the human intracranial tumors, gliomas and meningeomas. They
were able to characterize point to point spatial variations in normal
brain tissue and in intracranial tumors. In addition, they identified
homogeneities in tissue composition, and quantified the relative con-
centrations of proteins, lipids, and water in the brain tissue.