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W idefield Raman Imaging of Cells and T issues 159

to be useful in many applications, including work reported by
the Morris group at the University of Michigan, 20,21,22 measurement
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of chemical composition of plant tissue and investigation of
polymer crystallinity. 19
6.2.3 Other Modes of Generating Raman Images
While point and line mapping techniques are the most typical modes
of generating Raman images, other imaging methodologies reported
in the literature show promise for progress. Ma and Ben-Amotz
described a fiber-bundle image compression technique for acquiring
full Raman images with high spatial and spectral resolution in a short
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period of time. Schulmerich et al. demonstrated that laser power
distribution in such a technique not only eliminates thermal damage,
but also allows subsurface mapping of polymer samples beneath
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several millimeters of Teflon. In further work, Schulmerich et al.
developed a new system in which Raman maps were collected in
concentric circles by a circular fiber-optic array, also enabling the
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recovery of subsurface Raman data. In another variation of Raman
imaging, Ozaki’s group describe a Raman imaging probe in which
the images at different Raman shifts are achieved by tuning the
wavelength of a tunable laser for excitation. 27

6.2.4 Widefield Imaging
In widefield imaging, also known as chemical imaging, molecular
imaging, hyperspectral imaging, direct imaging and global imaging,
the entire sample field of view is illuminated and analyzed simulta-
neously by recording an image at discrete wavenumber increments
through the imaging spectrometer. Collected light is measured as a
function of both location in the sample field of view and wavelength.
In the image domain, the recorded data contains spatial information
at each wavelength. The spectral domain describes the molecules
contained in each pixel within the field of view. As a result, the widefield
image contains both structural and compositional information. 28
A variety of technologies have been used to achieve widefield
Raman imaging. They include dielectric filters in conjunction with
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tunable lasers, rotating dielectric filters, acousto-optic tunable
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filters (AOTF) and liquid crystal tunable filters (LCTF). One of
the first reported widefield Raman images was collected using
AOTF technology. Treado and coworkers demonstrated this Raman
imaging approach using a commercially available AOTF to obtain
rapidly high-fidelity images to monitor lipid/peptide model systems 31
and identify inclusions in breast tissue. 33
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First to develop an LCTF with a spectral resolution of 7 cm –1
and patent the technology for acquisition of widefield Raman
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images, Treado’s group further applied LCTF instrumentation to
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collect widefield Raman images of chicken breast tissue. At the