Biomedical Engineering Reference
In-Depth Information
Owing to the nonlinear nature of harmonic generation, the SHG signal level is proportional to the
square of the applied laser intensity, and thus it occurs predominately at the focus of a microscope objec-
tive, where the peak power is sufficiently high. This aspect gives SHG microscopy the powerful capability
of intrinsic 3D sectioning and is shared by other nonlinear optical (NLO) microscopy methods that have
been developed almost concurrently with SHG microscopy. The most well-known modalities include
third-harmonic generation (THG), two-photon excited fluorescence (TPEF), sometimes referred to as
multiphoton excited fluorescence (MPEF), and coherent anti-Stokes Raman scattering (CARS) micro-
scopy [7]. They all fall into the overarching category of laser scanning microscopy and experimentally
they are similar in many ways such as point scanning/detection and image formation. However, they are
all based on distinct physical processes that lead to different contrast mechanisms. Their applications as
imaging approaches are most often complementary, even though they can be readily incorporated into
one multimodal imaging system due to their common methodology. In this chapter, we give a detailed
comparison of these imaging methods. At the end of this chapter, some new developments of such non-
linear microscopy will be briefly reviewed.
4.2 Physical origin and contrast Mechanism of SHG
and the other nLo Microscopies
When a dielectric medium is exposed to an oscillating electric field
E
, a charge oscillation within that
medium will then be induced. Moreover, this charge oscillation has the capability of launching a sec-
ondary optical wave under certain conditions. All the NLO microscopic methods mentioned earlier
involve the response of some biological material to an incident laser beam of high intensity, and emis-
sion of the induced new optical wave that carries signatures of the imaged material. In this section, we
will discuss the nature of nonlinear interaction of light and matter that gives rise to the contrast mecha-
nism for each of the NLO imaging techniques.
The material response to the applied electric field
E
is often described using polarization
P
according
to the following relationship:
3
…
P
=
χ
( )
1
E
1
+
χ
( )
2
E
2
+
χ
( )
3
E
+
(4.1)
where χ
(
n)
is the
n
th-order nonlinear susceptibility. The nonlinear effects are represented by the higher-
order susceptibility (
n
> 1). he tensor χ
(
n)
decreases dramatically with increasing
n
, signifying that
higher-order nonlinear processes are very weak responses to the driving optical waves. This also
explains why NLO microscopy has only emerged as a standard tool in recent years, as the technology
requires user-friendly high-repetition-rate ultrafast lasers, which only became commercially available
in the mid-1990s. The first-order susceptibility, χ
(1)
, describing the linear response of the material to the
optical field, is often invoked to explain linear (one-photon) absorption and the index of refraction. SHG
is governed by χ
(2)
along with two closely related nonlinear processes, sum frequency generation (SFG)
and difference frequency generation (DFG). The third-order susceptibility, χ
(3)
, gives rise to THG, two-
photon and three-photon absorption, CARS, and stimulated Raman scattering (SRS). The readers are
referred to Refs. [8,9] for detailed discussion of all these different high-order susceptibilities.
4.2.1 SHG Photophysics
4.2.1.1 Molecular and Bulk Relationships
For SHG imaging, photon emission at the harmonic frequency 2ω
is collected as a result of second-order
nonlinear interaction of the fundamental optical wave (ω) and the nonlinear material. SHG is closely
related to hyper-Rayleigh scattering (HRS), an incoherent second-order light scattering process generat-
ing a new wavelength at 2ω in isotropic bulk solutions with random molecular orientation. In a sense,
