Scattering and absorption properties modification of optically cleared skeletal muscles: an ex vivo study

Optical clearing is a relatively new approach to enhancing the optical transparency of biological tissues by reducing their scattering properties. The optical clearing effect is achievable via various chemical, physical, and photo-thermal techniques. The present work studied optical parameters of bovine skeletal muscles under different clearing protocols: immersion optical clearing in 99% glycerol and photo-thermal optical clearing via exposure to IR laser irradiation. Moreover, the two techniques were combined with different immersion time intervals after multiple exposure periods to get optimum results. The muscle samples' diffuse reflectance and total transmittance were measured using a single integrating sphere and introduced to the Kubleka-Munk mathematical model to determine the absorption and reduced scattering coefficients. Results revealed a 6% scattering reduction after irradiating the sample for 10 min and immersing it in glycerol for 18 min and 8% after 20 min of laser irradiation and 18 min of immersion. Moreover, increases of 6.5% and 7.5% in penetration depth were prominent for the total treatment times of 28 min and 38 min, respectively. Furthermore, the measurements' accuracy and sensitivity were analyzed and evaluated using the receiver operating characteristic method. The accuracy ranged from 0.93 to 0.98, with sensitivity from 0.93 to 0.99 for each clearing protocol. Although laser irradiation and application of 99% glycerol separately produced scattering light reduction, the maximal clearing effect was obtained while irradiating the sample with a laser for 20 min and then immersing it in 99% glycerol for a maximum of 18 min.