Biomedical Engineering Reference
In-Depth Information
the target to be in a bulk noncentrosymmetric microenvironment, THG does not have this constraint.
However, THG is not highly versatile as a change in refractive index is required when focused beams
are used. This is because the signal arises from optical heterogeneities in the specimen where there are
changes in the third-order nonlinear susceptibility, χ
(3)
. One point of comparison comes from imag-
ing biological membranes with both SHG and THG. SHG images the two-dimensional membrane
itself where dye molecules have assembled parallel to each other [16,21,57]. In contrast, THG does so
by probing the volume around the membrane, where there is an interfacial change of susceptibility/
refractive index [32,49]. The sensitivity of THG to membrane boundaries has been successfully uti-
lized to image unstained whole zebrafish embryos with micrometer 3D resolution. This was used for
cell lineage reconstruction over the first 10 cell division cycles, with SHG complementary imaging of
mitotic spindles [49]. THG microscopy was also used to image lipid bodies in cells and tissues in real
time [32]. The examples indicate that THG, like SHG, is a relatively noninvasive microscopic method
that can be applied for
in vivo
imaging with 3D capability. However, the applicability is limited to
regions where these is a change in refractive index, and imaging bulk homogeneous tissues is not
possible.
4.4.4 cARS, SRS, and SHG comparison
Like SHG and THG, CARS and SRS are also coherent processes, where the generated signal maintains
a specific phase relationship with the incident waves. CARS and SRS are not limited by the noncentro-
symmetry requirement of SHG microscopy. In fact, CARS and SRS differentiate target tissues according
to their molecular vibrational signatures, and in principle, they have tremendous potential for biomedi-
cal imaging, as biological molecules (protein, DNA, lipid, etc.) all have distinct vibration spectra. This
has been increasingly enabled by the availability of widely tunable pico/femtosecond lasers with dif-
ference in photon energy that can be tuned to match the vibrational frequencies of target molecules.
For example, the versatility of label-free CARS and SRS microscopy has been demonstrated through
imaging of DNA backbone by targeting the PO
−
symmetric stretching vibrational mode [58], of plant
cell walls using the 1600 cm
−1
aryl ring stretching of lignin polymers [28], of omega-3 fatty acids, DMSO,
and retinoic acid as demonstrations of drug delivery monitoring [27,59], and of elastin and collagen
fibrils in Yorkshire pig carotid artery walls [60].
However, at the current stage of development, CARS and SRS are mostly used to image samples rich
in lipids using the 2845 cm
−1
CH
2
stretch mode. Examples have included lipid multilamellar vesicles
[61], endothelium cells of carotid artery [60], lipid droplets in mouse adrenal cortical cells [62], mouse
brain and ear skin [27], and the axonal myelin sheath from Guinea pigs [52,63]. This limitation is
in part due to weak Raman cross section and the resulting high number of local oscillators that are
needed to produce useful contrast. Moreover, the overall signal must be high enough to overcome the
TABLE 4.1
Comparison of Nonlinear Optical Microscopy Modalities
Primarily
Information
Emission
Direction
Detection
Wavelength
Method
Lasers
Concentration
Applicability
SHG
Structural/
assembly
Fs
Ti:sapphire
Quadratic
F/B
Tissue dependent
Bulk tissues
Near
UV-visible
THG
Structural/
assembly
OPO,
Cr:fosterite
Quadratic
F/B
Tissue dependent
Interfaces in
cells, tissue
Near UV
CARS
Chemical,
mostly C-H lipids
PS + OPO
Quadratic
Primarily
forward
Cells, tissues
Visible
SRS
Chemical,
mostly C-H lipids
Linear
Forward (laser
detection)
Cells, tissues
NIR pump
or probe
PS/fs + OPO
Two-photon
Protein
localization
Fs
Ti:sapphire
Linear
4π
Cells, tissues
Visible
