The application of SHG to the analysis of pathological conditions in muscle has lagged behind compared to applications using the collagen signal. We find, however, that muscle SHG is relatively simple to record using turnkey confocal systems, and that it provides, especially in combination with 2PEF, detailed information on the type and extent of muscle fiber damage. Sample handling is minimal and easy and the ability to image muscle fibers in their original surroundings is a clear advantage. SHG and 2PEF can detect all the critical changes in Pompe muscle fibers of both patients and mouse model. Additionally, these methods allow the complete and unbiased scanning of samples, which may reveal previously unnoticed information. It would be extremely easy, for example, to overlook the infrequent autophagic areas in some infant muscle biopsies.
SHG and 2PEF images of unstained muscle are complementary and reinforce each other. 2PEF can provide specific information about mitochondria, redox state of the muscle, and lysosomal degradation products such as lipofuscin. 2PEF is also useful for revealing the outline of the muscle as in the fragile infant biopsies (). While examination of SHG alone might suggest a hole in the tissue at the periphery of the fiber, 2PEF shows continuity with the neighboring fibers. Both pattern and intensity of the SHG signal convey structural information not easily accessible by other techniques and suggest previously unnoticed defects in PD muscles. For example, SHG shows that the myosin filaments of some PD muscle fibers are wavy, in contrast to the normal-looking ones in the next fiber (). Since waviness is a known characteristic of muscle fibers that have lost the ability to contract elastically (Brown et al., 1984
), these images suggest a failure to contract due to the buildup of non-contractile material and the atrophy of the fiber. 2PEF alone is relatively normal and would not reveal the damage. Work with single fibers might not reveal it either since an investigator may intentionally leave out wavy fibers, attributing this morphology to poor handling prior to fixation. SHG also shows that the stairlike transitions found in normal muscle (Both et al., 2004
) are present at higher frequency in PD muscles and that there are areas of poor organization in otherwise normal-looking areas of adult human PD fibers () in which 2PEF did not show any accumulation of autofluorescent material. Intensity of the SHG signal is related to the semi-crystalline order in muscle A-bands (Greenhalgh et al., 2007
). The local loss of SHG signal in infant biopsies, therefore, could reflect loss of sarcomeric organization. The highly variable sarcomeric length in the SHG images of biopsies from young infants () may be due to incomplete development as well as to the disease. The lack of biopsies from age- and origin-matched human infant controls makes it difficult at this point to evaluate the significance of these changes. It will be interesting to determine which of these defects are also present in other muscle pathologies.
SHG images show a better delineation of individual fibers than 2PEF. This is related more to cellular organization than to optics. Muscle fibers, especially type I, have layers of mitochondria next to the plasmalemma. The layers of two neighboring fibers are close enough to be unresolvable in diffraction-limited 2PEF. In contrast, the myofibrils from neighboring fibers are separated from each other by two such layers and are resolved in SHG.
The ease of sample preparation and the high information content of SHG and 2PEF combined should be useful in the observation and analysis of other muscle diseases, in particular those that involve displacement of organelles, such as the centronuclear myopathies (Pierson et al., 2005
) or formation of inclusions such as the tubular aggregates found in some myopathies (Chevessier et al., 2005
). The key to in vivo
applications is the possibility to record the backscattered SHG signal from muscle (Rothstein et al., 2006
). The signal is weak and a complicating factor is that it results in part from backscattered SHG and in part from the reflection of the forward signal (Légaré et al., 2007
). SHG, however, is still in its infancy. Not so long ago, nuclear magnetic resonance was a useful technique to study protein samples in thin glass tubes. Who would have predicted, then, the current use of MRI on alert humans? Likewise, it is highly probable that technical advances will make SHG more readily accessible in the future.