ARUP; ISBN: 978-0-9562121-5-3 - CMBBE 2012 - Cardiff University

measuring strain at different levels suggest different values of local strain compared to
the global applied strain. Screen et al [4] proposed a method to study local strain
distribution within tendon based on microscopic images and image analysis tool. Cell
nuclei are highlighted with fluorescent dyes and used as markers for strain analysis.
Figure 1 the hierarchal arrangement of tendon adapted from [5, 6]
A major problem with the investigations proposed in the literature is that the strain
measurement has been carried using 2D image processing techniques and thus cannot
accurately detect out of plane displacement information. This introduces error in strain
measurements as the sampling points move out of field of view due to twisting or
stretching of tendon during global tension.
3. EXPERIMENTS
For experiments, fascicles were taken from the distal end of the tails of male Wistar rats
aged between 2 months and 4 months. Fascicles were approximately 200µm in diameter
and were dissected from the tails within 1 hour of sacrifice. Prior to straining, fascicles
were incubated for 40 mins in 5 mM Acridine orange in PBS to stain the tenocyte
nuclei. Specimens were then rinsed in PBS prior to loading into the multiphoton
straining rig. The orientation of each sample was checked under brightfield settings
before the start of each strain regime.
A custom designed tensile straining rig, able to load fascicles and image them
simultaneously on the stage of a dipping lens Zeiss LSM 510 confocal fluorescence
microscope equipped with Coherent Chameleon lasers suitable for multiphoton
imaging. Fascicles were secured in one grip and allowed to hang under their own weight
while the second grip was tightened, creating a test length of 15 mm and defining the
0% strain position.
The rig containing the tendon fascicle was then immersed in PBS, and the rig placed on
the microscopic stage of the microscope. 10X magnification dipping objective lens was
used and the Chameleon laser was set to a wavelength of 900nm. This enabled the
imaging of the cells, labelled with Acridine orange, and at the same time, the collagen
via the second harmonic. Z stacked images were taken of each fascicle up to a depth of