Biomedical Engineering Reference
In-Depth Information
found to be equivalent for equal-biaxial stretch of materials with planar distributions.
For other loading conditions and fibre orientations, it was recommended that
κ
be
substantially less 0.1 to ensure errors in the GST model are less than 10 %.
6.3.5 Microstructural analysis
In this section, we discuss methods that have recently been developed to non-
destructively evaluate collagen and elastin fibre orientation simultaneous with uni-
axial loading experiments.
Multiphoton Microscopy (MPM) - 2PE and SHG
Using multiphoton microscopy, elastin can be imaged due to its intrinsic fluores-
cence under two- photon excitation (2-PE). The same microscope can be used to
simultaneously image collagen using its second harmonic generation (SHG).
Two photon excitation (2PE) microscopy
is so named because of the employment
of two-photon excitation. Briefly, two photons excite a flourophore in the test sam-
ple, resulting in the emission of a fluorescence photon. The wavelength of the emitted
photon depends on the type of fluorophore. In 2PE MPM, the emitted photons from
laser scanned surfaces are collected in a photomultiplier tube to generate images
which can then be 3D reconstructed. Compared with confocal microscopy, 2PE mi-
croscopy has deeper tissue penetration (up to 1 mm) more efficient light detection
and reduced phototoxicity. Elastin is well imaged by 2PE excited intrinsic fluores-
cence without exogenous stains [121].
Second harmonic imaging (SHG) microscopy
makes use of a nonlinear optical
phenomena in which photons interacting with a nonlinear material are combined,
forming new photons with twice the energy (so twice the frequency and half the
wavelength). The variation in contrast in the image arises due to the variability in
the specimen's ability to form second harmonic light. In particular, SHG enables
direct imaging of some anisotropic biological structures such as collagen without
staining or fixation [24]. As for 2PE microscopy, infrared light can be used, making
it possible to generate 3D images of specimen regions relatively deep in the tissue.
Uniaxial Multi-Photon Microscopy system (UA-MPM)
A uniaxial mechanical testing device was custom designed to operate in conjunction
with a multiphoton microscope (MPM) for coincident stress-strain analysis and laser
scan imaging of biotissues, Fig. 6.4, [49, 50]. During testing, arterial segments are
uniaxially loaded in the device under applied strain. Utilizing the second harmonic
generation (SHG) and two-photon excited autofluorescence (TPEA), endogenous
fibrillar collagen and elastin fibres are visualized. Image stacks are then 3D recon-
structed (Metamorph, Danaher, Washington, DC, USA and Imaris software, Bit-
plane, Switzerland).
In the UA-MPM system, data acquisition and motion control is performed with
National Instruments (NI, Austin, TX, USA) software and hardware. A stepper mo-
