Plotnikov Sergey, Juneja Vaibhav, Isaacson Ariel B, Mohler William A, Campagnola Paul J
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Biophys J. 2006 Jan 1;90(1):328-39. doi: 10.1529/biophysj.105.066944. Epub 2005 Oct 7.
Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.
利用二次谐波产生（SHG）成像显微镜，我们研究了用甘油进行光学透明处理对提高骨骼肌组织标本穿透深度的影响。我们发现，用50%甘油处理可使可实现的SHG成像深度增加2.5倍。使用快速傅里叶变换和连续小波变换进行的信号处理分析定量显示，肌节结构的周期性在透明处理过程中未发生改变，并且组织深处的图像质量通过透明处理得到了改善。SHG角偏振依赖性的比较也表明，肌动蛋白-肌球蛋白复合物的超分子组织没有变化。相比之下，对小鼠肌腱（基于胶原蛋白）进行相同处理导致SHG响应大幅下降。我们认为，甘油处理使肌肉实现光学透明的主要机制是细胞质蛋白浓度降低以及随之而来的对SHG信号的二次内滤效应减弱。成像深度对甘油浓度缺乏依赖性表明，折射率匹配在肌肉的光学透明中仅起次要作用。SHG和光学透明可能为研究高度散射的骨骼肌或心肌组织中的生理学提供一种理想机制，其穿透深度和可实现的成像深度都有显著提高。
