Cross‐Sectional Area Measurement Techniques of Soft Tissue: A Literature Review

Evaluation of the biomechanical properties of soft tissues by measuring the stress–strain relationships has been the focus of numerous investigations. The accuracy of stress depends, in part, upon the determination of the cross‐sectional area (CSA). However, the complex geometry and pliability of soft tissues, especially ligaments and tendons, make it difficult to obtain accurate CSA, and the development of CSA measurement methods of soft tissues continues. Early attempts to determine the CSA of soft tissues include gravimetric method, geometric approximation technique, area micrometer method, and microtomy technique. Since 1990, a series of new methods have emerged, including medical imaging techniques (e.g. magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound imaging (USI)), laser techniques (e.g. the laser micrometer method, the linear laser scanner (LLS) technique, and the laser reflection system (LRS) method), molding techniques, and three‐dimensional (3D) scanning techniques.


Introduction
T he accurate assessment of the cross-sectional area (CSA) of soft tissue (including tendons and ligaments) is a crucial prerequisite factor for estimation of biomechanical properties, such as Young's modulus, stress, elastic modulus, and energy density 1,2 . Many techniques to measure the CSA of biological samples have been presented in the literature.
Historically, the methods for CSA measurement included the gravimetric method, the geometric approximation technique, the area micrometer method, and the microtomy technique. The gravimetric method, which calculates the CSA by dividing the specimen's volume (obtained from its weight) by the length, was frequently used in the 1960s. Some authors used dried specimens 3 , while others used wet specimens [4][5][6] . The geometric approximation technique assumes that the CSA of the tendon is rectangular 7-13 , round [14][15][16] , or elliptical [17][18][19][20][21][22] , and measures the dimensions of the tendons by a micrometer or a microscope with a calibrated eyepiece micrometer. However, these methods, while convenient and non-destructive, may introduce inaccuracies and are not suitable for soft tissue with non-uniform shape. The area micrometer method allows CSA measurement of soft tissues with non-uniform shape by compressing specimens into a slot of known width and then measuring the heights of the sections [23][24][25] . However, it has been proved to cause permanent damage to tendons 26 . When compared with more accurate methods, both the geometric approximation technique and the area micrometer method underestimate the CSA of most ligaments by approximately 15%-40% 27 . The microtomy technique can obtain shape information as well as the CSA by cutting fresh specimens [28][29][30] or frozen specimens [31][32][33] into sections and then projecting or digitalizing the cross-sections. However, it is destructive and does not allow for subsequent biomechanical testing. Ellis compared seven CSA measurement methods for tendons 24 and found that the gravimetric method obtained the best repeatability for dried specimens, and that the gravimetric method and the area micrometer method were comparable in terms of repeatability for fresh specimens.
Since 1990, a series of non-destructive methods have emerged, including medical imaging techniques, laser techniques (e.g. the laser micrometer method, the linear laser scanner technique, and the laser reflection system), molding techniques, and three-dimensional (3D) scanning techniques.
Some researchers have attempted to measure soft tissue CSA using medical imaging techniques, such as magnetic resonance imaging (MRI) 34 , computed tomography (CT), and ultrasound imaging (USI) 35 . However, they faced problems of precision in small areas. Molding techniques determine the CSA of soft tissues by making casts of soft tissues using liquid-silicon/polymethylmethacrylate (PMMA) 26,27 or alginate 36,37 and measuring their cast directly. Laser techniques use a laser beam to access the width or radius information and then reconstruct the cross-sectional shape to access the CSA. Although the laser micrometer method is nondestructive and accurate (<2%), and can obtain the relevant geometry information, it is unable to detect concavities and requires all-round visibility 38,39 . The laser reflection system (LRS) can detect cavities and is the first device that could measure changing CSA during tensile testing, but the strain rate must remain slow (approximately 2 mm/min) 40 . 3D scanning techniques access the CSA by sectioning the 3D model of soft tissues acquired by optical, laser, or ultrasound techniques, such as 3D ScanTop, structured light scanning (SLS), or 3D freehand ultrasound. Molding, laser, and 3D scan techniques can obtain the geometric information of soft tissues.
This review summarizes the principles and reliability of CSA techniques and indicates where they are most applicable. Five databases were searched (including PubMed, Embase, Cnki, Wanfang, and Vip databases) using an agreed set of keywords. Studies investigating the CSA techniques of soft tissue were eligible. After filtering by two authors, 119 studies were included. The comparison of all CSA measurement techniques is shown in Table 1. Information on the included studies is presented in Table 2.

Methods
F ive databases were searched, including PubMed, Embase, Cnki, Wanfang, and Vip databases. The strategy had three components: soft tissue, CSA, and measurement. Two independent reviewers assessed the potential studies retrieved by EndNote, with any disagreements mediated by a third reviewer. Once duplicates were removed, titles, abstracts, and full texts of the studies were screened for eligibility according to the following inclusion criteria: (i) studies that were published as full reports before December 2019; (ii) studies that investigated the CSA measurement techniques of soft tissues; and (iii) studies that investigated the CSA measurement of airways, nerves, and muscle fibers. Finally, 119 studies were included in this review.

Gravimetric Method
T he gravimetric method calculates the CSA of soft tissues by dividing the specimen's volumeby the length. Volume can be determined by weight and density, specific gravity, or liquid displacement.
The gravimetric method was frequently used in the 1960s for CSA measurement of moist specimens 4-6 and dried specimens 3 . Ellis 24 compared four gravimetric methods (including moist specimen weight per unit length, moist specimen displacement volume per unit length, dry specimen weight per unit length, and dry specimen displacement volume per unit length) with area micrometer, shadow amplitude contour reconstruction, and planimeter measurements on photomicrographs of histological sections. Results showed that dry specimen weight per unit length had the best repeatability on single measurements for CSA of tendon specimens, and moist specimen gravimetric measurement and the area micrometer method were significantly more repeatable than shadow amplitude contour reconstruction.
This method made a considerable contribution in the early days: it can provide an approximate CSA and it is repeatable. However, errors are inevitably introduced. In addition, it is unreliable to determine the volume of soft tissues by water displacement, density, and weight. Cronkite (1936) found that this method did not provide consistent results 28 .
Haut and Little assumed the cross-sectional shape of the canine anterior cruciate ligament (ACL) to be elliptical and calculated the CSA by measuring the major and minor axes with a micrometer 17 . Woo et.al assumed the crosssectional shape to be rectangular and developed a micrometer instrument that applied a minimal compressive force to the specimen for thickness determination (Fig. 1), while the widths of specimens were measured with a cathetometer 8 . Matsumoto compared the CSA of rabbit Achilles' tendons measured by micrometer with CSA measured by observing tendon sections under a microscope, and found that the results did not differ 10 .
The geometric approximation technique is repeatable, convenient, non-destructive, and can be performed quickly. It can be applied for both fresh and dried tissues. However, it may introduce non-negligible inaccuracies and is not suitable for soft tissue with complex and non-uniform shape or concave surfaces. Micrometers or Calipers may compress specimens, resulting in overestimation.

Area Micrometer Method
T he area micrometer method determines the CSA of soft tissues by squashing them into a channel of known section until completely filled up and then measuring the heights of the sections by means of a micrometer head mounted upon the instrument.
The area micrometer method was first developed by Walker et al. (1964) and was applied to measure the CSA of human tendons 23 . It consisted of a rectangular slotted plate with a movable following bar. Three instruments with Allow morphometric measurements and suitable for almost all cross-sectional geometry. However, it is destructive, and residual fat and bisection process may cause overestimation. Methyl blue penetration and crystallized water evaporation will reduce contrast.

Wet
In vivo/in vitro

No
Exploit the spin density information in the sample to image.
Great contrast between tissues and image plane orientation can be set accurately and with 3D sequences. Allow multiple measurements. Can obtain morphological information, However, the relationship between the contrast in the MRI image corresponds with the actual borders of the tendon remains unknown and the equipment is expensive.

Ultrasonography Wet
In vivo/in vitro

No
Emit and receive reflected ultrasonic waves and processing the signal to produce an image. Ultrasonography, in particular brightness mode (Bmode) ultrasound, has been widely used.
It is readily available, relatively inexpensive, nondestructive, and its temporal resolution is good, allowing for dynamic imaging and fast measurements. Portable devices are also available. Can obtain morphological information and allow repeated measurements.
However, it needs expensive equipment and samples need to be immersed in a saline water bath, introducing the possibility of swelling. It needs all-around visibility and reflection occurring at the boundary between two media may result in poor reliability to observe the borders.

Wet
In vivo/in vitro No X-ray radiological imaging technique which yields transverse tomographic images reflecting the spatial distribution of X-ray attenuation in the part examined.
Easy, rapid, accurate, and commonly used diagnostic techniques. It can clearly define the boundary at the interface between fat and muscle. However, it is quite a complex and expensive technique not adaptable to all kinds of tests. Non-destructive, can obtain geometry information, and allow multiple measurement.
However, the concave region is assumed to be flat. It needs all-round visibility and the data acquisition process is relatively slow. The operations are complicated.

Laser reflection system (LRS)
Dry/wet

In vitro
No Rotate laser sensor around the soft tissue samples in a circular path and then reconstruct the cross-sectional shape and determine cross-sectional area by the inner radius (r) of the specimen relative to the rotating center and corresponding angle in polar coordinates using Simpson's rule.  Cervical spine ligaments Particular cross-section was projected on a paper and an outline of the ligament boundary was traced and analyzed using a computer-aided design (CAD) program.  Gastrocnemius medialis different slot width are fabricated to permit measurements of tendons of a wide range of CSA. Results showed that the area micrometer had great reproducibility and the error was less than 1%. To better control the pressure to reduce errors caused by pressure, the constant pressure area micrometer was invented by Ellis et al. 24 , which could squash the tissue with a constant force and read CSA from a suitably calibrated linear variable differential transformer (LVDT) or dial gauge (shown in Fig. 2A). In the operation, the specimen was placed between a pair of stainless-steel side blocks which were positioned 0.0307 inch apart. In 2003, an area micrometer with an oval-shaped slot was customized 49 (shown in Fig. 2B). Specimens are placed into the oval-shaped slot and a constant pressure of 0.12 MPa is applied with an attached spring. The sliding caliper is read and then the CSA is calculated from this displacement using the following formula: CSA = 3 × 3 × 3.14 + 6 d. Pressure is a key factor for the area micrometer. Sufficient pressure must be applied to prevent overestimation, but excessive pressure may cause damage to tissues. With the increase of press time, tissue specimens will be stiffer due to the loss of liquid/ground substance 26 . Standard values of 0.12 MPa applied pressure and 2 min deformation time are ideal 25 .
In conclusion, the area micrometer method allows the measurement of CSA of fresh soft tissue with non-uniform shape. It has great reproducibility 24 and is easy to use. The instrument is relatively affordable, portable, and already commercialized. Although there are many more accurate alternatives, the area micrometer method is still popular and widely used. However, it has several obvious shortcomings. This technique is also unable to obtain shape information  and cannot be applied to dried. specimens Moreover, the area micrometer method consistently compresses specimens with pressure and confines them to a specific shape. Therefore, it has been proved to cause permanent damage to tendons. 26 When compared with more accurate methods, both the geometric approximation technique and the area micrometer method overestimate the CSA of most ligaments by approximately 15%-40% 27 .

Microtomy Technique
T he microtomy technique determines CSA by staining and sectioning specimens for digitization. Some researchers section fresh specimens, while others section flash-frozen specimens, which is called the freezefracture (Cryomicrotomy) technique. Liquid nitrogen is usually used as the freezing agent. Soft tissue is coated with a methyl blue dye to enhance contrast during imaging.
In 1936, Cronkite presented a microtomy method that obtained the CSA of fresh tendons by cutting sections 0.5-mm to 1.0-mm thick from tendon specimens with a thin razor blade, projected shadows of them, and measured CSA with a planimeter 28 . He compared this technique with methods that obtain CSA by dividing volume with length and found the section method to be more accurate. In 2014, Iriuchishima measured the anterior cruciate ligament (ACL) mid-substance CSA by sectioning fresh ACL, as shown in Fig. 3 51 . However, without freezing, it is hard to obtain uniform sections and to cut sections perpendicular to the long axis of specimens.
Mkandawire later developed a freeze-fracture technique for measuring ligament CSA to determine human foot and ankle ligament morphometry, in which ligaments were quickfrozen with an immersion technique using isopentane chilled by a liquid nitrogen bath 33 . Isopentane has great stability at subzero temperatures and is inert to human tissue. Then, ligaments were bisected by hammer and ground wood chisel. Methyl blue and liquid paper were used for enhancing contrast. The cryomicrotomy technique has been applied to obtain the geometric and mechanical properties of human cervical spine ligaments and lumbar spinal ligaments 31,50 . In Buchanan's research, tendon specimens were embedded in Tissue-Tek OCT compound and quick frozen in liquid nitrogen 32 .
On the one hand, the cryomicrotomy technique allows for morphometric measurements of soft tissues and is better adapted to measurement of complex cross-sectional geometry, such as short ligaments (less than 10 mm) and ligaments with bony projections. On the other hand, the cryomicrotomy technique is destructive and does not allow for subsequent biomechanical testing and multiple measurements 33 . In addition, residual fat may cause overestimation. Methyl blue penetration and crystallized water vaporized during the immersion process will reduce contrast. Shearing generated during the ligament bisection may result in overestimation.

Medical Imaging Techniques
S ome researchers have attempted to measure soft tissue CSA using medical imaging techniques, such as MRI and ultrasound. All three medical imaging techniques can be used for measuring CSA in vivo.

Magnetic Resonance Imaging
The MRI technique exploits the spin density information in the sample to image; therefore, it is completely noninvasive 105 . It has long been used to qualify changes in muscle volume and CSA after exercise. In 2001, Anderson et al. used MRI technology to measure the CSA of the anterior cruciate ligament ACL 34 . However, they assumed the CSA of soft tissue was elliptical and MRI fails to capture the details of the boundary due to resolution limitations. In addition, rotation during imaging will change the shape and dimensions of soft tissues, so it would be difficult to make sure soft tissue always line up with the imaging plane and, thus, obtain an oblique section. Stenroth suggested that MRI provides superior reliability for tendon CSA measurements compared with USI 63 . MRI has been A B suggested to be the gold standard for measuring the tendon CSA to investigate the validity of USI-based methods 69,106 . However, Couppe noted that tendon CSA measured by MRI is associated with an underestimation compared with the molding technique, but by optimizing the measurement using a 3 Tesla MRI and the appropriate National Institutes of Health color scale, this underestimation could be reduced to 2.8% 55 . MRI CSA measurement can also be used to predict the expected ACL autograft size 41 . Figure 4 shows the MRI image for hamstring tendon CSA measurement, which traces the CSA using of the region-of-interest tool.
Magnetic resonance imaging provides great contrast between tissues and there is no pressure applied on the tissue. Image plane orientation can be set accurately, and with 3-D sequences, the imaging plane can be adjusted postimaging (re-slicing) 63,107 . However, how well the contrast in the MRI image corresponds with the actual borders of the tendon remains unknown, and, as a result, the MRI-based measurement may either underestimate or overestimate the tendon CSA.

Ultrasound Imaging
Ultrasonic imaging involves emitting and receiving reflected ultrasonic waves and processing the signal to produce an image. The higher the frequency, the weaker the diffraction and the higher the image resolution. However, high frequency may also result in poor penetration. Ultrasonography, in particular brightness mode (B-mode) ultrasound, has been widely used to quantify the CSA and to investigate the morphologic and mechanical properties of soft tissues in vivo and in vitro 101 .
Diagnosing tendinopathy often involves the measurement of tendon size using diagnostic USI. Brushoj et al. and Richards et al. investigated the reliability of MRI and diagnostic USI in CSA measurement of Achilles and tibialis tendons and results indicated high intra rater-and inter-rater reliability between USI and MRI 65, 108 . Galanis et al. found that the ICC among the USI, MRI, and intraoperative graft methods for the semitendinosus tendon and the GT data ranged from 0.502 to 0.906 70 . Figure 5 shows the USI of the semitendinosus and the gracilis tendon CSA measurement. In 2002, Noguchi et al. designed an in vitro ultrasonographybased system to determine the CSA of the tendon and found that there is no significant difference between the use of ultrasonography and digital calipers, which implies that ultrasonography would also underestimate the CSA 35 , the schematic of which is shown in Fig. 6. Ultrasound imaging is an appealing method of choice for scientific research and clinical evaluation. It is readily available, relatively inexpensive, and non-destructive, and its temporal resolution is good, allowing for dynamic imaging and fast measurements. Portable devices are also available. This method can also obtain morphological information of the sample section and can be repeated at will without damage to the tissues. However, expensive equipment is required and samples need to be immersed in a saline water bath, introducing the possibility of swelling. It has also proved to be inaccurate 69 . Furthermore, bone needs to be resected to provide clear vision. Moreover, reflection occurs at the boundary between two media with different acoustic densities, which may result in poor reliability to observe the borders and, thus, cause overestimation or underestimation of CSA.

Computed Tomography
Computed tomography scanning is an X-ray radiological imaging technique that yields transverse tomographic images reflecting with high accuracy the spatial distribution of X-ray attenuation in the part examined 109 and gives an exact and accurate cross-sectional picture of the soft tissues 110 . X-ray attenuation is indicative of the type of tissue under scan and can, therefore, be used to construct an image of the internal structure of the body 77 . Because of the high-density resolution and collimation system of CT, it can distinguish small differences in soft tissues without the interference of the extra-lamellar structure. CT is mainly used for CSA measurement of thigh muscle 75,[78][79][80] . The CT image of the left thigh for CSA measurement is shown in Fig. 7.
With the rapid development of the CT scanning technique, fast image acquisition and high spatial resolution images were realized. The use of new medical imaging software made it possible to measure areas within specified attenuation limits. Peripheral quantitative CT (pQCT) is commonly used for soft tissue area quantification by segmenting regions representing different tissues. Sherk compared human muscle and fat CSA measurements between MRI scans and pQCT and found that CSA did not differ significantly between MRI and strongly filtered pQCT images 79 . Haggmark's research shows that CT is an accurate way of measuring the size of different muscle bellies 75 . Clement et al. demonstrated that reliable and accurate measurements of the articular surfaces can be measured using a freeform tool on CT scans 111 . Diffusible iodine-based contrastenhanced CT (diceCT) and μCT, which includes both iodine-based staining and the use of other staining agents, was developed recently as a means of visualizing muscle portions in situ, which enabled high-resolution data to be collected 103 .
Computed tomography and new medical imaging software enable easy and rapid assessment of the CSA of soft tissues. These are commonly used diagnostic techniques and can provide an exact and accurate cross-sectional picture of the soft tissues. CT scans provide much clearer information than plain X-rays 112 and a CT scan is also expected to be more accurate than ultrasound scanning because it can clearly define the boundary at the interface between fat and muscle 113 . However, it is quite a complex and expensive technique that is not adaptable to all kinds of tests.

Molding Techniques
M olding techniques measure the CSA of soft tissues by making a cast of soft tissues and measuring their cast directly. In this way, accurate CSA and morphological information including concavities of soft tissue can be accessed with no damage to the soft tissue. According to molding materials, molding techniques can be divided into the silicone rubber/PMMA molding technique 26,27,81,82 and the alginate molding technique 36,37,[83][84][85]106 , examples of which are shown in Fig. 8.
The silicone rubber/PMMA molding technique was developed by Race and Amis in 1996. They determined the CSA of soft tissue by making a silicone rubber cast and a PMMA replica of a specimen 26 . Then the PMMA replica was sliced and stained, and the CSA was calculated by square counting. A curing agent was mixed with liquid silicone rubber to speed up the curing process. However, due to the PMMA shrinkage and square counting method, there was a systematic underestimation of 6.2% and a random error of 1.8% after correction. The problem of PMMA shrinkage was solved by optimizing molding materials using a formula by Schmidt et al. 27 . The application of camera and Image analysis software greatly improved the measurement accuracy, to 2%. The silicone rubber/PMMA molding technique is accurate, non-destructive, allows measurement of non-uniform shape specimens, and can obtain morphological information. There are still a few potential limitations. It is time-consuming and soft tissue needs to be exposed to air for 2 h while molding, which may cause dehydration. If moisturizing tissue with saline, excess saline could gather and create bubbles in the mold and, thus, change the CSA. All of this reduces the success rate of casting.
The alginate molding technique uses dental alginate impression materials as molding materials 36 . After the molding process, alginate mold was sliced and photographed. Results show that the alginate molding technique has high measurement accuracy (0.8%) and a short operation time  (approximately 5 min). The alginate mold is easy to cut so a thin blade can be used to obtain a flat cut and the shrinkage is almost 0% within 10 min. Casting is not needed. It can accurately measure an area of 3 mm 2 , which is smaller than for the silicone rubber molding method (7 mm 2 ). However, this technique cannot be applied to measuring the CSA of more complex soft tissue (such as the anterior cruciate ligament) or soft tissue with bony ends that are hard to pull out of the mold.

Laser Micrometers
Laser micrometers can obtain the cross-sectional shape and area of soft tissues by reconstructing the cross-sectional shape based on the width measurements. The widths of specimens are obtained using collimated laser beams. In 1988, Lee and Woo developed a non-contact CSA measurement technology based on laser micrometers (shown in Figs 9 and 10) 38 . During width measurement, specimens were placed perpendicular to collimated laser beams and rotated 180 . Then, the specimen's cross-sectional shape was reconstructed based on width measurements following a specially designed algorithm, and the CSA was calculated using Simpson's rule. This study compared the CSA of eight porcine ACL specimens measured by laser micrometers and the area micrometer method, and revealed that the results obtained by area micrometer method was 17% lower than those obtained by laser micrometers. In 1990, Woo made the acquisition process for the laser micrometer system automated and fully computerized, which sped up the process. He applied this method to determine the cross-sectional shape of a rabbit medial collateral ligament and ACL and the results showed that the measurement accuracy of laser micrometer was less than 2% 39,114 . In Harner's study, specimens are placed perpendicular to the ground, which can decrease the deformation of specimens during rotation 115 . Race and Amis found, compared to the replica method, that the laser micrometer method overestimate CSA by an average of 2.3 and 1.5% (SD) for tendons 26 . In 2010, Bruneau developed an optic micrometer that submerges the tendon into a measuring compartment filled with cold saline solution and estimated the CSA of rat tail tendons within a 2% margin of error 97 . overhead view of the CCD laser reflectance system with a biological specimen over the center of rotation (COR) of the system. "R" represents the total radius of the system, "d" represents the distance to the surface of the specimen, and "r" represents the inner radius of the specimen with its respective angle (y) 90 .
Laser micrometer is non-destructive, can obtain the geometric information, and allows multiple measurement. However, the laser micrometer is affected by specimen geometry. The concave region is assumed to be flat during the image reconstruction and, thus, overestimated the CSA by 19% compared to the casting method 26 . Moreover, it needs allround visibility and, therefore, the removal of adjacent bones is required, which has negative impacts on implantation. The data acquisition process requires approximately 1-2 min for each cross-section to collect numerous data points. There are also systematic errors associated with increments, and the operations are complicated and expensive.

Laser Reflection System
The Laser Reflection System (LRS) measures the CSA of soft tissues by rotating the laser sensor around the soft tissue samples in a circular path. The cross-sectional shape is reconstructed and the CSA is determined by the inner radius (r) of the specimen relative to the rotating center and the corresponding angle in polar coordinates using Simpson's rule.
In 1995, Chan developed a laser reflectance system using position-sensing detector (PSD) laser displacement sensors 116 . PSD are more sensitive to surface properties (i.e. hydration and opacity) than charge-coupled device (CCD) laser displacement sensors. With the development of CCD, in 2006, Moon developed and validated a CCD laser reflectance system composed of a CCD laser sensor and rotary motion table (shown in Fig. 12) 90 and found that the accuracy of the system was less than or equal to 2.0% with a repeatability of 0.0%. The cross-sectional shapes obtained with this system were in good agreement with those obtained by laser micrometer and the laser micrometer system overestimated CSA by approximately 6%. Favata improved the CSA measurement method for small connective tissues by using a laser triangulation sensor in combination with two LVDT to acquire the thickness and x and y displacements 91 . This technique has been used in animal 92,93 and human tendons 94,117 . Pokhai developed a new laser reflectance system capable of measuring changing CSA of soft tissues during A B  tensile testing (shown in Fig. 13), which was designed to be installed on an Instron 8872 servohydraulic test machine; the measurement accuracy of this system was less than 4.3% 40 . It has been applied to the CSA measurement of rat tendons 93 and human forearm tendons 94 .
In conclusion, the LRS can successfully measure the CSA of soft tissues with concavities in an accurate, repeatable, and rapid manner (shown in Fig. 11). It is a non-contact, non-destructive, and accurate tool for CSA measurement and is the first device that could measure changing CSA during tensile testing. However, it is too slow to use during mechanical testing because a complete revolution of the LRS takes over 20 s to complete and the strain rate must be slow (approximately 2 mm/min), which may introduce error caused by the viscoelastic of soft tissues 40 . The rotation center should be in the cross-section of the specimen. Another limitation of the CCD laser sensor is that it does not perform as well for semitransparent surfaces, so the tissues need to be stained. The accuracy was affected by specimen size as well as the spot beam diameter. Therefore, only specimens larger than 20 mm 2 were considered currently 90 . Moreover, when the angle between laser beams and the target surface is steep, the artifact of the shape of the cross-section is generated due to the light reflecting far from the charge-coupled device CCD sensor 95 .

Linear Laser Scanner
The linear laser scanner (LLS) is composed of two CCD laser reflectance devices mounted facing each other on two carts sliding on horizontal and parallel linear guides, with the specimen placed vertically between the two horizontal guides, the schematic of which is shown in Fig. 13 95 . During measurement, the lasers are moved along the guides to sweep the specimen in the x−y plane and measure the distance from the laser to the specimen surface. Then image reconstruction is processed to obtain a cross-sectional shape and the CSA is calculated using MATLAB.
In 2010, Vergari et al. designed an LLS which could obtain accurate and repeatable CSA (less than 2% error) with short acquisition time (within 2 s per measurement) and, thus, could perform CSA measurements under continuous tension 95 . The LLS has been used to measure the CSA of equine superficial digital flexor tendons 96 .
The LLS Linear Laser Scanner is accurate, repeatable, and fast and easy to assemble and operate. It can adapt to various types of testing machines and is capable of moving to follow a defined zone on the specimen during testing. The system does not need precise centering of the sample and can perform noncontact measures during mechanical testing. However, this device cannot precisely acquire steep concavities due to the reflection of the laser beams. The specimen maximum measurable thickness (45 mm) is also a limitation.

Three-Dimensional Scanning Techniques
T hree-dimensional scanning techniques access the CSA by sectioning the 3D model of soft tissues acquired by optical, laser, or ultrasound techniques, such as SLS and 3D freehand ultrasound.
The optical 3D scan system was developed by Hashemi et al. using a commercially available photographic scanner, 3D Scantop (Olympus America), to construct the 3D image of a human ACL 98 . This system is accurate, easy to apply, and affordable, at US$5000. It also allows the determination of CSA at any position of the tissues and all relevant information can be extracted from one single application of the method. However, as with most optical techniques, the system cannot detect surface concavities.
Freehand 3D ultrasound (FUS) combines brightness mode (B-mode) USI with a motion analysis system to generate 3D reconstructions of anatomic structures in vivo 100,101,118 .
Reconstructed 3D volume is created using a rigid body calibration method to transform sequential 2D images into a global coordinate system, which is subsequently used for object segmentation. Three-dimensional ultrasound has been validated to have excellent repeatability 99 and can provide reliable measurements 101 . However, accurate 3D reconstructions can only be achieved when ultrasound images are obtained during static conditions and the accuracy of results may not be representative of the accuracy of measurements in vivo 101 . Figure 14 shows the application of FUS on Achilles tendon CSA measurement.
Structured light scanning can reconstruct the 3D digital models of soft tissue using a variation on stereophotogrammetry. Hayes et al. developed a CSA measurement technique using a commercially available structured light scanner named Artec Spider and integrating it with a custom mechanical rig permitting 360 acquisition of the morphology of soft tissues 102 . The reconstructed 3D model was then used to measure the CSA of the tendon. Specimens should be light coated with flour to reduce excessive reflection. This technique can measure the entire shape without contact but long-term reliability and the cost of the device may be potential limitations.
Other techniques, such as the 3-D Scantop imaging system 98 , the 3 T scanner (Signa PET/MR,GE Healthcare) 104 , the three-dimensional CT 112 (3DCT: three-dimensional gray-scale-images) that may be viewed from different angles and in real-time rotation), can also be used to obtain 3D images and compute the CSA.