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Raman Imaging for Biomedical Applications in Clinics 267

Chandrasekhara V. Raman the idea that this phenomenon owed its
origin to the scattering of sunlight by the molecules of the water. After
his return to Calcutta, he started investigations with his student
Krishnan. In 1922, Raman published his first observations, ideas, and
physical concepts as Molecular Diffraction of Light. In April 1923, he
experimentally showed that “associated with the Rayleigh-Einstein
type of molecular scattering, there was another and still feebler type
of secondary radiation, the intensity of which was of the order of
magnitude of a few hundredths of the classical scattering, and dif-
fered from it in not having the same wavelength as the primary or
incident radiation.” Many experiments followed, and later, in 1928
Raman developed the theory, that they observed the optical analogue of
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the Compton effect. At the same time several other laboratories around
the world (Rayleigh, Robert Wood, Landsberg, and Mandelstam) were
investigating the same subject. The Russian scientist Mandelstam
reported the effect in crystalline quartz and calcite, and called that
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inelastic light-scattering phenomenon “combinatorial scattering.” In
1930, Raman won the physics Nobel Prize for his work on the scatter-
ing of light and for the discovery of the Raman effect.
In 1975, Delhaye and Dhamelincourt introduced the first “Raman
microprobe” or “Raman microscope” and outlined several approaches
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to Raman imaging. 14,15 In the last three decades, with further devel-
opment of lasers and detectors, the technology became much more
sensitive and interest in using Raman spectroscopy in studies of com-
plex biological systems and in biomedical applications strongly
increased, leading to the first applications in cell and tissue studies. 17
The first Raman-based intracellular results were obtained in 1990–1991
when Puppels et al. developed a highly sensitive confocal Raman
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spectrometer enabling high-resolution single-cell studies. Nowa-
days numerous applications of Raman spectroscopy on cells and tis-
sues have been developed, both in vitro and in vivo. 19–24

9.1.2 Principles
Light can interact with atoms and molecules in different ways. Pho-
tons can be absorbed (in some cases followed by emission of another
photon, like in fluorescence), or they can be elastically or inelastically
scattered, as can be depicted in the Jablonski diagram of Fig. 9.1. In
the elastic scattering process there is no energy transfer between the
light and the scattering molecules. The wavelength of the scattered
light has the same frequency as the incoming light. This scattering
process is known as Rayleigh scattering.
If the photon is inelastically scattered by the molecule, some energy
is transferred from the photon to the molecule or vice versa. This energy
is used to increase or decrease the vibrational energy of the molecule
and the wavelength of the scattered light is different from the incident
light. This scattering process is known as Raman scattering. If the
vibrational energy of a molecule is increased, the scattered photon