Biomedical Engineering Reference
In-Depth Information
diagnosis of corneal pathologies. In this chapter, we will discuss the principles and instrumentation of
multiphoton microscopy that is relevant to corneal imaging, and present results that demonstrate prom-
ises of multiphoton imaging in corneal diagnostic imaging.
12.2 Principles of Second Harmonic Generation Microscopy
The recent development of second harmonic generation microscopy (SHGM) has led to the exciting
possibilities for pathological diagnostics. Like multiphoton fluorescence excitation, SHG is a nonlinear
process offering similar advantages such as optical sectioning capability, improved penetration depth,
label-free imaging, and significantly reduced photo-damage [45,54,55]. In SHGM, a near-infrared,
ultrafast laser is chosen as the excitation source since it can provide light beam with high, instantaneous
intensity below the tissue destruction threshold. Furthermore, the femtosecond source is effective in
generating the nonlinear signal (multiphoton-excited fluorescence and SHG) within the focal volume
[52,56]. In addition, the noncentrosymmetric structure of stromal and scleral collagen is a strong gen-
erator of SHG signal. Therefore, SHGM is an ideal technique for imaging the intrinsic triple-helical,
noncentrosymmetric structures of cornea and sclera collagen fibril without extrinsic labeling [53,57,58].
12.2.1 Second Harmonic Generation Microscopy
Higher harmonic generation is a nonlinear polarization process related to the interaction of intense
light with matters. In general, the polarization,
P
i
, of a material can be expressed as
P
=
χ
E
+
χ
E E
+
χ
E E E
+
(12.1)
i
ij
j
ijk
j
k
ijkl
j
k
l
where χ
ij
, χ
ijk
, and χ
ijkl
are, respectively, the first-, second-, and third-order susceptibility tensors, and
E
is the applied electric field amplitude. The second term of this expression χ
ijk
E
j
E
k
represents the induced
SHG polarization, which contributes to the generation of radiation field at one half wavelength of the
excitation source. Thus, in the SHG process, two near-infrared incident photons are converted into one
visible photon at twice the energy. As a result, there is no energy deposited in the illuminated specimen
during the SHG process. This further allows microscopic imaging to be achieved with minimal invasion
[12]. In addition, when the electric field is reversed, it corresponds to the reversal of polarization direc-
tion but does not include the second- and even higher-order terms because the SHG signal depends on
the square of the electric field. As a result, the second harmonic signal can only be produced in noncen-
tral symmetric material, such as structures composed of collagen molecules.
12.2.2 Multiphoton Microscopy instrumentation
A typical multiphoton imaging system that can be utilized for corneal imaging is shown in Figure
12.1. For excitation, a titanium-sapphire (ti-sa) laser (Tsunami, Spectra Physics, Mountain View, CA)
pumped by a diode-pumped, solid-state (DPSS) laser system (Millennia X, Spectra Physics) is used.
The 780 nm output wavelength of the ti-sa laser is used for sample excitation and can be guided into
a commercial upright microscope (E800, Nikon, Japan) by a pair of galvanometer-driven, x-y mirrors
(Model 6220, Cambridge Technology, Cambridge, MA). After entering the microscope, the laser light
is beam-expanded and reflected by the short-pass, primary dichroic mirror (700 dcspruv-3p, Chroma
Technology, Rockingham, VT) onto the back aperture of the focusing objective (such as S Flour, 40 × /
NA 0.8, water-immersion WI, Nikon). Multiphoton images can then be acquired by scanning the focal
spot across the specimen. To obtain three-dimensional images at both high resolution and large scale,
a motorized stage (H101, Prior Scientific, UK) is adopted to the microscope for specimen translation
after each optical scan. After signal collection by the focusing objective in the epi-illuminated geometry,
