Design and development of non-contact method
for measurement of cross-sectional area of soft
tissues during tensile test
Tam Zhi Yang
U055551U
Bioengineering 4
Introduction
Ligament and tendon are passive tissues and are responsible for the structural integrity of the
musculoskeletal system by transferring forces between muscle and bone or from one bone to another
[1]. To carry out this function properly, it is important that their mechanical property is well suited to a
specific application. In the study of mechanical and material properties of the ligament and tendon,
cross sectional area is often required in the calculation of the mechanical properties [1], hence the
importance in accurately estimating the cross sectional area. Traditionally, measurement of the cross
sectional area of soft tissue is conducted using contact based method, such as casting technique,
thickness calipers and micrometer [2]. The method mentioned involved contact with the specimen
during measurement, and the applied forces may force the fluid out of the tissue and result in
deformation of the cross sectional profile [3]. In view of the short coming that comes with contact based
methods, non-contact method for measurement of cross sectional area is desirable. Non contact
methods fall into three categories: Medical imaging techniques (CT, MRI), profile width and profile
radius techniques [4]. Profile width technique reconstruct the cross sectional profile by measuring the
width of the soft tissue at different angle. The data obtained is then used to re-create the cross sectional
profile by back projection (e.g. Woo and others, [3]). Profile radius re-create cross sectional area profile
of the sample by measuring the radius at different angle. In this study, a laser system for the
measurement of complex cross sectional area during tensile test was developed. Methods developed
(e.g. profile radius, profile width) requires the sample to rotate 3600 to collect the necessary data point
for cross sectional profile reconstruction. Depending on the size of the system, a 3600 rotation might not
be feasible in most cases due to the configuration of the tensile testing machine due to obstruction by
parts of the tensile testing machine. Therefore, alternate method that is able to collect the necessary
data point with a smaller rotation angle is developed.
Method and material
Laser system
A pair of laser displacement sensor (LK-G30, Keyence, United Kingdom) was used in this study. The lasers
were arranged in opposite of each other with the limit of detection of both the laser overlapping (figure
1). Object that is placed outside the limit detection (not touching the line) would not be detected by one
of the laser, and hence the width of the soft tissue on that particular plane could not be estimated. The
laser sensors were secured on to the rack with cable tie. To ensure that the laser pairs are parallel to
each other, guides were installed at the edge of the rack (figure 3). The rack is mounted on x-y tables
which allow the rack to move in the x-y direction. The x-y table is then mounted on an Instron machine.
Laser
Rack
X
Laser
Laser
Beam
Limit of
detection
Y
Figure 1. Position of laser and line for the limit of detection
Grip
Laser displacement Sensor
Leveler
Figure 2. Laser displacement sensor on top of a supporting rack. To ensure that the rack is parallel to the
ground, a leveler is used.
Sample
Guide
Figure 3. Guide installed at the edge of the supporting rack to aid in the parallel position of the laser
sensor.
Z-Axis
X-Y table
X-Axis
Y-Axis
Figure 4. Laser rack mounted on a X-Y table.
To circumvent the need to rotate 3600 to obtain the necessary data point for reconstruction,
information of the width of the sample along the x-axis is collected by moving the laser system along xaxis at an increment of 0.01mm. Once the scan along x-axis was completed, the data was then
processed on a spreadsheet program (Excel 2007, Microsoft Corporation, United States) and the profile
was reconstructed (figure 5). As seen from figure 5, the end of the profile could not be captured by the
laser sensor. This might be due to the fact that the laser beam did not reflect back to the sensor. This
also lead to the possibility that the data close to the ends might not be accurate, hence 10% of the data
points at the end were not included in the reconstruction. In order to create a complete profile of the
cross sectional area and obtain information on the ends of the cross sectional area, another scan was
performed 90 degree from the first scan. The profiles from these two scans were then superimposed to
create a complete cross sectional profile (figure 6).
3
2
1
0
-1 0
2
4
-2
-3
Figure 5. Cross sectional profile created from one scan.
3
2
1
0
-1 0
2
4
6
-2
-3
Figure 6. Cross sectional profile created from two perpendicular scans.
Since the laser system was not originally designed to rotate, the plate that connects the laser to the
Instron machine has to be redesigned. In this new design, instead of using one plate to connect the laser
rack and the Instron, two plates will be used. Between these two plates, set of holes that are 90 degree
apart will be drilled and the two plates will be connected to each other through these two holes. By
connecting the plates through different sets of hole, the laser system can be rotated to obtain cross
sectional profile that is perpendicular to each other. With the complete cross sectional profile, the cross
sectional area of the sample can be estimated using ImageJ.
Figure 7. Plates with set of holes that allow the whole laser system to rotate.
During the data processing step, a factor is introduced to calibrate the data. Since the laser sensor is
very sensitive (capable of detecting a difference in width up to 0.001mm), it is impossible to adjust the
laser in such a manner than the two laser are exactly 5.000mm apart, in which the limit of detection of
both the laser overlap exactly. Hence, before each measurement begins, a calibration run on a cylinder
was conducted. The data from the calibration run was then used to adjust the data from the ligament.
Preparation of ligament for histology analysis
Porcine ligament was used to compare the cross sectional area estimated by the laser system with the
cross sectional area estimated by histological analysis. The porcine ligament was fixed in 10% buffered
formalin for 1 week and then processed in a graded series of ethanol before clearing in toluene. The
process ligament was subsequently embedded in paraffin and sectioned until the region, previously
marked for laser-scanning, was observed. Images of this region were taken and used for area
measurement via ImageJ.
Results and Discussion
Cylinder
Measurement of the cross sectional area of a cylinder with known area was conducted.
In the first experiment (table 1), measurement was conducted on different section of the cylinder to
determine if the CSA measurements differ along the length of the sample. If it does it would mean that
the sample was not sufficiently vertical and might have introduced errors larger than other factors
combined. The results suggest that the sample could be placed with enough verticality to ensure that it
does not introduce a significant error to the measurement with the help of a pendulum. Data that was
calibrated showed significant improvement in accuracy and precision (table 2 and table 3). Though the
calibration factor was small (e.g. the adjustment to the data is in the range of 0.01mm), the effect this
has on the area measurement is significant because the accumulation of error inherent in the method of
cross sectional area measurement. This also justify the use of a calibration factor because it will be
challenging to adjust the relative position of the laser by 0.01mm.
The error ranged from -0.68% to 1.44% which is smaller than the laser reflectance system by Moon et al.
for cross sectional area of 23.1mm2 (Average error of -2%) [2]. It is also slightly smaller than the imagebased method by Salisbury et al. (Maximum error 1.69%) for a cross sectional area of 16.3mm2[4].
Table 1. CSA measurement along the cylinder length
CSA measurement from micrometer (mm2)
CSA from laser sensor
Upper part
Middle part
Lower part
Arbitrary
cross
section
1st cross
section
2nd cross
section
11.824
11.970
12.079
12.022
Error
1.88%
2.16%
1.68%
Table 2. CSA measurement with no data calibration
CSA from
1st measurement
2nd measurement on same
2
micrometer (mm )
cross section
11.824
CSA from laser
Error
CSA from laser
Error
sensor (mm2)
sensor (mm2)
12.047
1.88%
12.083
2.19%
12.304
4.06%
12.164
2.88%
Arbitrary
cross section
1st cross
section
2nd cross
section
3rd cross
section
4th cross
section
Table 3. CSA measurement with data calibrated
CSA from
1st measurement
2nd measurement on same
micrometer (mm2)
cross section
11.824
CSA from laser
Error
CSA from laser
Error
sensor (mm2)
sensor (mm2)
11.756
-0.57%
11.977
1.3%
11.743
-0.68%
12.014
1.6%
11.837
0.11%
11.696
-1.11%
11.872
0.41%
11.994
1.44%
Average error
0.31±0.01%
Porcine ligament
Measurement of a porcine ligament was also conducted and the result compared to measurement
obtained by histological method. The cross sectional area of the ligament based on data obtained from
the laser sensor is about 20.74mm2, compared to 15.29mm2 estimated by the histological method. Due
to the irregularity of the morphology of the ligament, part of the ligament was not captured (red circle
in figure 4) and line was used to breach to gap. The inability of the laser to capture a complete profile in
this case is due to the size of the ligament. The laser will fail to reflect back to the CCD sensor if the
specimen comes too close to the sensor.
The discrepancy in the data might be due to the dehydration process involved and may have resulted in
the shrinkage of the ligament. Hence to better access the accuracy of the laser system in estimating the
cross sectional area, other method that does not change the morphology of the ligament should be
attempted, such as measurement by micro-CT.
Figure 4. Reconstructed Cross sectional profile of the ligament from data obtained from laser sensor.
The red circle cover the edge where no data was obtained from laser sensor.
Figure 5. Cross sectional profile of the porcine ligament analyzed by the laser system prior to histology
analysis
Conclusion
The laser system developed has proved to be successful in estimating the cross sectional area of object
with regular shape (e.g. cylinder). However, at the time of this report was written, its efficacy on sample
with irregular object has not been proven. For specimen that is smaller and is less irregular in shape than
the porcine specimen, it is likely that the laser system would be able to provide a reasonable estimation
of the cross sectional area.
Reference
1. Goodship AE, Birch, HL. Cross sectional area measurement of tendon and ligament in vitro: a
simple, rapid, non-destructive technique. Journal of Biomechanics 38(2005), pp. 605-8.
2. Moon, DK, Abramowitch SD, Woo, SL. The development and validation of a charge-coupled
device laser reflectance system to measure the complex cross-sectional shape and area of the
soft tissue. Journal of Biomechanics 39 (2006), pp. 3071-5.
3. Woo SL, Danto MI, Ohland KJ, Lee TQ, Newton PO. The use of a laser micrometer system to
determine the cross-sectional shape and area of ligaments: a comparative study with two
existing methods. J Biomech Eng. 1990 Nov;112(4):426-31.
4. Salisbury ST, Buckley CP, Zavatsky, AB. Image-based non-contact method to measure crosssectional areas and shapes of tendons and ligaments. Measurement science and technology 19
(2008) 045705.