Quantitative second harmonic generation imaging and modeling of the optical clearing mechanism in striated muscle and tendon

Abstract

We have investigated the mechanisms and capabilities of optical clearing in conjunction with second harmonic generation (SHG) imaging in tendon and striated muscle. Our approach combines three-dimensional (3-D) SHG imaging of the axial attenuation and directional response with Monte Carlo simulation (based on measured bulk optical properties) of the creation intensity and propagation through the tissues. Through these experiments and simulations, we show that reduction of the primary filter following glycerol treatment dominates the axial attenuation response in both muscle and tendon. However, these disparate tissue types are shown to clear through different mechanisms of the glycerol-tissue interaction. In the acellular tendon, glycerol application reduces scattering by both index matching as well as increasing the interfibril separation. This results in an overall enhancement of the 3-D SHG intensity, where good agreement is found between experiment and simulation. Through analysis of the axial response as a function of glycerol concentration in striated muscle, we conclude that the mechanism in this tissue arises from matching of the refractive index of the cytoplasm of the muscle cells with that of the surrounding higher-index collagenous perimysium. We further show that the proportional decrease in the scattering coefficient mu(s) with increasing glycerol fraction can be well-approximated by Mie theory.

Optical clearing of biological tissues: prospects of application in medical diagnostics and phototherapy

“…The amplification of the signal was also due to better packing of the fibrils. It was also shown that the axial attenuation of the direct SHG signal is reduced with the increase of the glycerol concentration (25, 50, and 75%) [72]. The backward SHG signal considerably decreased in the process of optical clearing due to the scattering reduction and the change of the local density of dipoles, producing the second harmonic [72].…”

“…Optical clearing of the mouse tail tendon in vitro exposed to the glycerol solutions with different concentration was demonstrated in the papers by LaComb et al [72], Nadiarnykh and Campagnola [73], and Rylander et al [31]. The authors managed to increase the probing depth in the second harmonic generation microscopy up to 450 µm at the expense of considerable reduction of the scattering coefficient (up to 130 folds at the wavelength 890 nm during 5 hours) and increasing the packaging density of fibrils (according to the data of electron microscopy, the volume fraction of fibrils increased from 0.65 to 0.9) [31].…”

“…New original results obtained using the combination of optical clearing with the known optical visualisation methods, such as laser speckle-contrast imaging [34, 71,75,77,79], OCT [20,29,35,[59][60][61]65,69,80,82], microscopic imaging [23,24,83,84], ultra-microscopy [85][86][87], etc., demonstrated high potentiality of their mutual use not only for getting high-resolution structure and functional tissue images in vitro [72,73,[83][84][85][86][87], but also for optical imaging and diagnostics in vivo [25,34,45,50,59,61,65,68,70,71,[75][76][77][79][80][81]. Table 1 shows the increase of the light penetration depth, caused by clearing agents in some diagnostic methods.…”

Quantification of glucose and glycerol diffusion in myocardium

“…3, 4, 11-17 and recent ones present optical clearing of tissues such as skin, [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] sclera, [33][34][35] and skeletal muscle. [36][37][38][39][40][41] We have found that only in two papers optical clearing of heart tissues were investigated. 42,43 Authors of Ref.…”

“…In spite of the more or less intensive studies of optical clearing e±ciency of skeletal muscle [36][37][38][39][40][41] and a few studies on cardiovascular muscle tissue, 42,43 di®usion coe±cients neither for glucose nor glycerol in the myocardium have not been measured yet. The knowledge of di®usion coe±cients for OCA-water°u xes in tissues allows one to understand the optical clearing mechanisms more precisely, to improve the mathematical models describing the interaction of the OCAs with the tissue.…”