Optical Imaging in Biology: Basics and Applications
653
7 Second Harmonic Imaging (SHG) Microscopy
Imaging-based on non-linear optical effects includes second harmonic generation
microscopy (SHG). It is a second-order coherent process where two lower-energy
photons are up-converted to exactly twice the incident frequency (half the wavelength) of an excitation laser. The second-harmonic generation was first observed
in birefringent crystals. Biological SHG imaging was first reported in 1986 when
Freund investigated the polarity of collagen fibers in rat-tail tendon at a resolution of
~50 μm [70]. In 2002, Mohler and Campagnola reported practical implementation
of tissue imaging at high resolution and rapid data acquisition using laser scanning, and since then, SHG microscopy has been an increasingly used imaging tool
[71]. Second-harmonic imaging provides information similar to classical histology
and electron microscopy with additional structural information in four dimensions
and without the need for complex and destructive sample preparation. Typical SHG
microscopy instrument requires a mode-locked femtosecond laser and a modified
scanning microscope (reflection or transmission). Tunable (700–1000 nm) titanium
sapphire oscillator is the most commonly utilized light source for SHG microscopy.
One of the primary requirement for a molecule to be visible by SHG microscopy is
that it should be non-centrosymmetric. SHG microscopy has been applied widely
to visualize the fibrillar distribution of collagen, particularly the collagen-I fibrils,
and study various diseases related to collagen disorder. One of primary medical
application of SHG microscopy is in identifying Fibrosis associated with multiple
internal organs such as kidney, liver and lung. SHG provides molecular structure
information and typically, the obtained information is complementary to TPEFM.
It also offers more penetration depth due to near-IR excitation and has relatively
lower photo toxicity effects. Schanne-Klein for the first time demonstrated potential
utility of SHG microscopy for quantifying collagen changes in mouse kidney disease
[72]. Using the density and the value of average brightness in microscopic images,
they observed an increase associated with the disease. Two-photon or multiphoton
excited harmonic generation microscopy has also enabled us to study extracellular
matrix, and bones without the use of labeling agents such as dyes. In addition to
fibrosis, collagen deposition (desmoplasia) is also known to be enhanced in certain
cancers. In Fig. 8, authors have shown that desmoplasia can serve as an effective and
early marker for breast cancers. They characterized three different tumor-associated
collagen signatures (TACS) that can be associated with the pathogenicity of the
disease [73].
8 Hyperspectral Imaging (HSI)
HSI combines traditional imaging with spectroscopy to collect both spatial and spectral information simultaneously [74]. Each of the pixels in a hyperspectral image has
one spectrum that can be used for characterization. A typical HSI system is consists
653
7 Second Harmonic Imaging (SHG) Microscopy
Imaging-based on non-linear optical effects includes second harmonic generation
microscopy (SHG). It is a second-order coherent process where two lower-energy
photons are up-converted to exactly twice the incident frequency (half the wavelength) of an excitation laser. The second-harmonic generation was first observed
in birefringent crystals. Biological SHG imaging was first reported in 1986 when
Freund investigated the polarity of collagen fibers in rat-tail tendon at a resolution of
~50 μm [70]. In 2002, Mohler and Campagnola reported practical implementation
of tissue imaging at high resolution and rapid data acquisition using laser scanning, and since then, SHG microscopy has been an increasingly used imaging tool
[71]. Second-harmonic imaging provides information similar to classical histology
and electron microscopy with additional structural information in four dimensions
and without the need for complex and destructive sample preparation. Typical SHG
microscopy instrument requires a mode-locked femtosecond laser and a modified
scanning microscope (reflection or transmission). Tunable (700–1000 nm) titanium
sapphire oscillator is the most commonly utilized light source for SHG microscopy.
One of the primary requirement for a molecule to be visible by SHG microscopy is
that it should be non-centrosymmetric. SHG microscopy has been applied widely
to visualize the fibrillar distribution of collagen, particularly the collagen-I fibrils,
and study various diseases related to collagen disorder. One of primary medical
application of SHG microscopy is in identifying Fibrosis associated with multiple
internal organs such as kidney, liver and lung. SHG provides molecular structure
information and typically, the obtained information is complementary to TPEFM.
It also offers more penetration depth due to near-IR excitation and has relatively
lower photo toxicity effects. Schanne-Klein for the first time demonstrated potential
utility of SHG microscopy for quantifying collagen changes in mouse kidney disease
[72]. Using the density and the value of average brightness in microscopic images,
they observed an increase associated with the disease. Two-photon or multiphoton
excited harmonic generation microscopy has also enabled us to study extracellular
matrix, and bones without the use of labeling agents such as dyes. In addition to
fibrosis, collagen deposition (desmoplasia) is also known to be enhanced in certain
cancers. In Fig. 8, authors have shown that desmoplasia can serve as an effective and
early marker for breast cancers. They characterized three different tumor-associated
collagen signatures (TACS) that can be associated with the pathogenicity of the
disease [73].
8 Hyperspectral Imaging (HSI)
HSI combines traditional imaging with spectroscopy to collect both spatial and spectral information simultaneously [74]. Each of the pixels in a hyperspectral image has
one spectrum that can be used for characterization. A typical HSI system is consists