We are investigating the use of optical coherence tomography (OCT) for examining muscle pathology, specifically the skeletal muscle tissue of mouse models of muscular dystrophy. This work is a collaborative effort with Professor Miranda Grounds’ Skeletal Muscle Research Group, and also Assoc/Prof Gavin Pinniger, both within the School of Anatomy, Physiology and Human Biology at The University of Western Australia.
Duchenne muscular dystrophy (DMD) is the most severe form of the human muscular dystrophies, characterised by the progressive degeneration of skeletal and cardiac muscle tissue. The disease is caused by a mutation of a recessive gene located on the X-chromosome that codes for the muscle membrane protein dystrophin, which is vital for preserving mechanical integrity of the muscle ﬁbres (myofibres). Absence of a functional form of this protein results in a fragile membrane that is easily damaged, activating an inﬂammatory response that leads to myofibre death (necrosis). The disease occurs at a rate of ~1 in 3600 to 6000 male births worldwide, and proves fatal due to respiratory or cardiac failure.
Mice animal models are routinely used in pre-clinical studies aimed at understanding the cause and progression of the disease, and assessing the effectiveness of potential therapies for DMD. Histology is conventionally used to study the morphology of skeletal muscle, but the process is laborious and time consuming, and not amenable to examining large tissue volumes. Instead, we are exploring the use of OCT as a medical imaging alternative. OCT is a minimally-invasive imaging modality capable of acquiring three-dimensional (3D) scans of biological tissue with a resolution of 1 – 20 µm at depths up to 1 ~ 2 µm.
We have investigated OCT imaging of muscle pathology using two mouse models:
- Whole muscle autograph (WMA) surgical model of skeletal muscle damage and inflammation ; and
- Mdx mouse model for dystropathology in human DMD. 
We have performed 3D-imaging of large volumes of ex vivo mouse skeletal muscle tissue to identify features of interest, such as: regions of healthy and/or intact myofibres; damaged and/or necrotic myofibres; inflammation and infiltrating inflammatory cells; connective and adipose tissue; and areas of pathology. This has been achieved using the following techniques:
- Three-dimensional OCT (3D-OCT) imaging, using both time-domain , ,  and spectral-domain , ,  OCT systems;
- Needle probes for OCT for deep-tissue imaging ; and
- Parametric imaging of:
Section 1. 1 3D-OCT imaging of muscle pathology
1) 1.1 Whole muscle autograft (WMA): Surgical model of muscle damage and inflammation 
We first investigated OCT imaging of muscle pathology, using the whole muscle autograft (WMA) . The WMA is a surgical model of skeletal muscle damage, inflammation and regeneration, which has been used extensively in the research to help understand the basic biology of muscle. Specially, it has been used to understand the progression of pathology in skeletal muscle, the mechanisms of regenerating new tissue following this damage, and the effect of pharmaceutical interventions on these processes of necrosis, inflammation and regeneration of skeletal muscle tissue. En face OCT images obtained from the 3D-OCT datasets were matched with subsequent transverse hematoxylin and eosin (H&E)-stained histology, corresponding for depth and visually correlating structural features of interest. In the OCT images of the WMA, the damaged and inflamed graft tissue of the extensor digitorum (EDL) skeletal muscle could be readily distinguished from the healthy and intact host tibialis anterior (TA) muscle, meeting at a clearly defined boundary. Regions of tissue breakdown and inflammation could be identified in the graft tissue, and myofibres seen in the underlying host tissue. The features identified in the OCT images matched well with the corresponding H&E-histology, as shown in Figure 1.1.
2) 1.2 Mdx mouse: Genetic model of human Duchenne muscular dystrophy 
We next investigated OCT imaging of muscle pathology in the treadmill-exercised X-chromosome-linked muscular dystrophy (mdx) mouse model of DMD. Our results have shown that 3D-OCT is capable of imaging the large-scale structure and pathology of ex vivo muscle samples from the mdx. Necrotic lesions (areas containing necrotic myofibres with fragmented membranes and spilled cytoplasm, cellular debris, infiltrating inflammatory cells, connective and adipose tissue) occur in the skeletal muscles in response to the exercise and underlying disease. Location of these necrotic lesions is aided by the injection of the marked Evans blue dye (EBD) into the mice immediately after treadmill-exercise, but 24 hours prior to sampling. The EBD-positive degenerating myoﬁbres can be macroscopically identiﬁed by their dark blue staining, which reveals the severity and spatial location of the muscle damage in the animal to help guide our 3D-OCT imaging. Necrotic lesions could be identified within these large volumes (mm3) by 3D-OCT based on the loss of intrinsic optical contrast, as seen in Figure 1.2.
We validated OCT images by comparing them with co-located conventional histology using two staining techniques: hematoxylin and eosin (H&E) and fluorescent Evans blue dye (EBD), as seen in Figure 1.3 (c) and (d) respectively. The striation pattern observed in the en face OCT images correspond to areas of intact myofibres, which can clearly be seen in the co-located H&E-histology sections as centrally nucleated (CN) regenerated myofibres, and the source of the OCT appearance is attributed to the presence of the regular alignment of intact myoﬁbres.
Regions of disrupted OCT signal are seen in the images as featureless areas corrupted by speckle that have lost the distinctive striation pattern, and are characterised by low signal and contrast within a relatively well-deﬁned boundary. These disruption regions (DR) are attributed to necrotic lesions (NL) and the underlying change in the muscle tissue structure as a result of dystropathology, as seen in the corresponding H&E-stained histology sections Figure 1.3 (c) and F2 below. EBD-positive degenerating myoﬁbers are also identified in the frozen histology sections by their red ﬂuorescence, indicating regions containing permeable (leaky or necrotic) myofibres that are often associated with necrotic lesions.
We have found that 3D-OCT is useful for the structural assessment of pathology and identiﬁcation of lesions in skeletal muscle tissue from the mdx mouse model of DMD. It is able to image the structural features of interest from skeletal muscle tissue (myoﬁbres, tendon, connective tissue, adipose tissue) based on the intrinsic differences in the scattering properties of these structures. It can identify muscle necrosis and lesions, which are seen in scans as regions of low signal, low contrast and a loss of the characteristic striation pattern of intact myoﬁbres. We have also shown that EBD is a suitable in vivo marker for targeted 3D-OCT imaging of necrotic lesions and muscle pathology, as it does not interfere with the OCT imaging beam at 1300 nm.
Section 2. 2 Needle probe for deep tissue OCT imaging of muscle pathology 
We have also developed an extremely miniaturised OCT needle probe (outer diameter 310 μm) with a high sensitivity (108 dB) that has enabled us to image the structure and pathology of the mdx mouse deep within its skeletal muscle . Details of this OCT needle probe are shown below in Figure 2.1.
Using the miniaturised OCT needle probe, we were able to acquire three-dimensional volumetric images from ex vivo mouse tissue, examining both healthy mucle from wild-type mice and pathological muscle from dystrophic mdx mice. An example of this is shown below in Figure 2.2.
Regions of intact myofibres were again seen as striations (Figure 2.3) whilst necrotic lesions were seen as a loss of these striations (Figure 2.4). We could also detect tendon and connective tissue deep within skeletal muscle tissue, based on their high backscattering. These observed structures were validated against co-registered hematoxylin and eosin (H&E) stained histology sections. This work has demonstrated the ability of using an OCT needle probe to visualise structure and pathology of at the microscopic level deep in skeletal muscle tissue in situ.
Section 3. 3 Parametric OCT imaging of muscle pathology
Extending on our initial work of imaging skeletal muscle pathology using 3D-OCT, we have investigated three different techniques. These methods offer improved contrast of muscle pathology by extracting additional parameters from the raw 3D-OCT data.
1) 3.1 Attenuation coefficient µt using three-dimensional optical coherence tomography (3D-OCT) 
We assessed muscle pathology in ex vivo tissue samples from dystrophic mdx mice, by quantitative parametric imaging of the local total attenuation coefficient μt using three-dimensional optical coherence tomography . An example of this is shown below Figure 3.1, where the parametric image of μt(x,y) improves the contrast between the muscle pathology (necrotic lesion) and the surrounding regions of intact myofibres.
The resulting values of the local total attenuation coefficient μt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice were found to be are higher (9.6 ± 0.3 mm-1) than regions from the same tissue containing only necrotic myofibres (7.0 ± 0.6 mm-1), and significantly higher than values from intact myofibres, whether from an adjacent region within the same sample (4.8 ± 0.3 mm-1) or from healthy muscle tissue of wild-type mice (3.9 ± 0.2 mm-1) used as a control. These results are summarised in the histogram of Figure 3.2 below. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy.
2) 3.2 Birefringence ∆n using polarisation-sensitive optical coherence tomography (PS-OCT) 
Undamaged muscle tissue possesses high levels of optical birefringence due to its anisotropic ultrastructure, and this birefringence decreases when the tissue undergoes necrosis. Using polarisation-sensitive optical coherence tomography (PS-OCT) scans, we are able to quantify the birefringence in muscle tissue, generating an image indicative of the tissue ultrastructure, with areas of abnormally low birefringence indicating necrosis. The technique additionally gives a measure of the proportion (volume fraction) of necrotic tissue within the three-dimensional imaging field of view. Comparing the percentage necrosis assessed by this technique against the percentage necrosis obtained from manual assessment of histological sections, we found the difference between the two methods to be comparable to the interobserver variability of the histological assessment. This is the first published demonstration of PS-OCT to provide automated assessment of muscle necrosis .
3) 3.3 Mechanical strain mε using optical coherence micro-elastography (OCME) 
In muscle pathology, such as that seen in human DMD, impairment of skeletal muscle function is closely linked to changes in the mechanical properties of the muscle constituents. Optical coherence micro-elastography (OCME) uses optical coherence tomography (OCT) imaging of tissue under a quasi-static, compressive mechanical load to map variations in tissue mechanical properties on the micro-scale. We have conducted the first study of OCME on skeletal muscle tissue, in both healthy wild-type mice and dystrophic mdx mice exhibiting muscle pathology. Our results have shown that OCME can resolve the important features of muscle tissue, including myofibres, fascicles (connective tissue surrounding a bundle of many myofibres) and tendon, and can also detect necrotic lesions present in skeletal muscles of mdx mice, as shown below Figure 3.4. In many instances, OCME provides better or additional contrast complementary to that provided by OCT. Our results suggest that OCME could provide new understanding and opportunity for assessment of skeletal muscle pathology .
Section 4. Key techniques
Section 5. Key researchers
- Blake R. Klyen, Julian J. Armstrong, Steven G. Adie, Hannah G. Radley, Miranda D. Grounds, David D. Sampson, “Three-dimensional optical coherence tomography of whole muscle autografts as a precursor to morphological assessment of muscular dystrophy in mice”, Journal of Biomedical Optics, 13(1), 011003 (2008).
- Blake R. Klyen, Thea Shavlakadze, Hannah G. Radley-Crabb, Miranda D. Grounds, David. D. Sampson, “Identiﬁcation of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography”, Journal of Biomedical Optics, 16(7), 076013 (2011).
- Xiaojie Yang, Dirk Lorenser, Robert A. McLaughlin, Rodney W. Kirk, Matthew Edmond, M. Cather Simpson, Miranda D. Grounds, and David D. Sampson, “Imaging deep skeletal muscle structure using a high-sensitivity ultrathin side-viewing optical coherence tomography needle probe”, Biomedical Optics Express, 5(1), pp. 136-148 (2014).
- Blake R. Klyen, Loretta Scolaro, Tea Shavlakadze, Miranda D. Grounds, and David D. Sampson, “Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient”, Biomedical Optics Express, 5(4), pp. 1217–1232 (2014).
- Xiaojie Yang, Lixin Chin, Blake R. Klyen, Tea Shavlakadze, Robert A. McLaughlin, Miranda D. Grounds, and David D. Sampson, “Quantitative assessment of muscle damage in the mdx mouse model of Duchenne muscular dystrophy using polarization-sensitive optical coherence tomography”, Journal of Applied Physiology, 115(9), pp. 1393-1401 (2013).
- Lixin Chin, Brendan F. Kennedy, Kelsey M. Kennedy, Philip Wijesinghe, Gavin J. Pinniger, Jessica R. Terrill, Robert A. McLaughlin and David D. Sampson, “Three-dimensional optical coherence micro-elastography of skeletal muscle tissue”, Biomedical Optics Express, 5(9), pp. 3090-3102, (2014).