Optical Imaging in Biology: Basics and Applications
651
But the enormous benefits offered by these methods are counterbalanced by the fact
they require an interaction between the external probe and the molecule of interest.
This can cause a change in the native conformation of the biomolecules and can
induce functional changes. As an alternate, label-free methods are being explored.
Here the imaging is performed by different photophysical processes employing basic
light-matter interaction principles in their native environment. The image contrast is
generated by differences in the biochemical composition and varying physiological
environment.
Raman spectroscopy named after its discoverer, Sir. C.V. Raman is based upon the
inelastic scattering of light [57]. In the year, 1966 Delhaye and Migeon for the first
time provided evidence that Raman scattering is independent of the sample volume
and can be applied through a microscope [58]. This hypothesis led to the development and commercialization of the first Raman microspectrometer. A typical Raman
microscope involves a combination of a spectrometer with a microscope with an
ability to provide both spatial and molecular composition specific information. The
output is often in the form of an image cube where spectral characteristic associated with each pixel is accumulated. Starting in 1950s, several variations of Raman
spectroscopy such as coherent anti-Stokes Raman spectroscopy (CARS), surfaceenhanced Raman spectroscopy (SERS), resonance Raman spectroscopy (RRS), tipenhanced Raman spectroscopy (TERS) and stimulated Raman spectroscopy (SRS)
aiming at enhanced sensitivity have been developed [59].
Among the different variants, SERS have received large attention of the scientific community as it offers extremely high sensitivity with respect to spontaneous
methods. The primary hypothesis of SERS is based on enhancing the signals through
plasmonic effects where molecules are adsorbed on a metallic surface leading to
enhancement by a factor up to 10
15 . Some of the recent studies have reported singlemolecule detection using SERS including in vivo biological imaging. In the references, some of the recent developments and diverse applications of SERS have been
summarized [60–62]. In TERS, The high sensitivity of SERS is coupled with the
spatial resolution of a scanning probe microscopy. A metalized tip (Au or Ag) is
placed in close contact (μm) with the substrate to create a Plasmon effect with
high resolution (nm) [63]. Similar to SERS this method has also been utilized for
different imaging applications ranging from material sciences to biology [63]. Resonance Raman helps in improving the overall selectivity of the technique. This involves
choosing an excitation wavelength closer to the absorption band of the target molecule
and thereby enhancing the signals by several orders of magnitude. Often lasers in
the ultraviolet range are used to minimize the fluorescence contamination. Additional details about this method are summarized well in a review article by Efremov
et al. [64]. CARS and SRS are the two recently identified non-linear approaches of
Raman imaging. In addition to high resolution, these methods also offer high speed
and therefore can be applied for in vivo imaging. At room temperature, most of
the photons stay in the ground state therefore CARS imaging involves excitation of
photons through two laser pulses [59, 65]. The first laser helps to excite the photons
to the first vibrational state and the second one assists in transferring this electron to
the virtual states. Similarly, in SRS two lasers are also used but the wavelengths are
651
But the enormous benefits offered by these methods are counterbalanced by the fact
they require an interaction between the external probe and the molecule of interest.
This can cause a change in the native conformation of the biomolecules and can
induce functional changes. As an alternate, label-free methods are being explored.
Here the imaging is performed by different photophysical processes employing basic
light-matter interaction principles in their native environment. The image contrast is
generated by differences in the biochemical composition and varying physiological
environment.
Raman spectroscopy named after its discoverer, Sir. C.V. Raman is based upon the
inelastic scattering of light [57]. In the year, 1966 Delhaye and Migeon for the first
time provided evidence that Raman scattering is independent of the sample volume
and can be applied through a microscope [58]. This hypothesis led to the development and commercialization of the first Raman microspectrometer. A typical Raman
microscope involves a combination of a spectrometer with a microscope with an
ability to provide both spatial and molecular composition specific information. The
output is often in the form of an image cube where spectral characteristic associated with each pixel is accumulated. Starting in 1950s, several variations of Raman
spectroscopy such as coherent anti-Stokes Raman spectroscopy (CARS), surfaceenhanced Raman spectroscopy (SERS), resonance Raman spectroscopy (RRS), tipenhanced Raman spectroscopy (TERS) and stimulated Raman spectroscopy (SRS)
aiming at enhanced sensitivity have been developed [59].
Among the different variants, SERS have received large attention of the scientific community as it offers extremely high sensitivity with respect to spontaneous
methods. The primary hypothesis of SERS is based on enhancing the signals through
plasmonic effects where molecules are adsorbed on a metallic surface leading to
enhancement by a factor up to 10
15 . Some of the recent studies have reported singlemolecule detection using SERS including in vivo biological imaging. In the references, some of the recent developments and diverse applications of SERS have been
summarized [60–62]. In TERS, The high sensitivity of SERS is coupled with the
spatial resolution of a scanning probe microscopy. A metalized tip (Au or Ag) is
placed in close contact (μm) with the substrate to create a Plasmon effect with
high resolution (nm) [63]. Similar to SERS this method has also been utilized for
different imaging applications ranging from material sciences to biology [63]. Resonance Raman helps in improving the overall selectivity of the technique. This involves
choosing an excitation wavelength closer to the absorption band of the target molecule
and thereby enhancing the signals by several orders of magnitude. Often lasers in
the ultraviolet range are used to minimize the fluorescence contamination. Additional details about this method are summarized well in a review article by Efremov
et al. [64]. CARS and SRS are the two recently identified non-linear approaches of
Raman imaging. In addition to high resolution, these methods also offer high speed
and therefore can be applied for in vivo imaging. At room temperature, most of
the photons stay in the ground state therefore CARS imaging involves excitation of
photons through two laser pulses [59, 65]. The first laser helps to excite the photons
to the first vibrational state and the second one assists in transferring this electron to
the virtual states. Similarly, in SRS two lasers are also used but the wavelengths are