Retention of polarization signatures in SHG microscopy of scattering tissues through optical clearing

Abstract

Polarization responses in Second Harmonic Generation (SHG) imaging microscopy are a valuable method to quantify aspects of tissue structure, and may be a means to differentiate normal and diseased tissues. Due to multiple scattering, the polarization data is lost in turbid tissues. Here we investigate if this information can be retained through the use of optical clearing which greatly reduces the scattering coefficient and increases the corresponding mean free path. To this end, we have measured the SHG intensity as a function of laser polarization and the SHG signal anisotropy in murine tendon and striated muscle over a depth range of 200 microns. We find that the laser polarization is highly randomized in the uncleared tissues at depths corresponding to only 2–3 scattering collisions (50-10 microns). This depolarization of the laser is also reflected in the randomized anisotropy of the SHG signal as it is created over a range of polarization states. In strong contrast, both polarization signatures are significantly retained through ~200 microns of tissue thickness following treatment with 50% glycerol. Moreover, the measured polarization responses for both tendon and striated muscle are consistent with the extent of reduction of the respective scattering coefficients upon clearing. We suggest the method will be applicable to SHG imaging of connective disorders as well as cancer through several hundred microns of extracellular matrix.

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

“…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.…”

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space

“…As polarization is scrambled in scattering tissues [31], the tissues were optically cleared in 50% glycerol/PBS overnight [31]. Giant unilamellar vesicles (GUVs) were created from L-aPhosphatidylcholine lipid (Avanti Polar Lipids) for circular polarization calibration.…”

“…As the tendon is sufficiently thick to support multiple scattering events which randomize the polarization response, the specimen must be first optically cleared to reduce the scattering and increase the transparency [31]. Here we define 0° condition as where the laser polarization is aligned with the long axis of a collagen fiber(s).…”

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.…”