Influence of laser beam aberrations compensation and spot size on the transmittance in native and optically cleared skeletal muscles

Light propagation and penetration inside tissues highly affect the optical imaging of biological tissues. Two major factors influence that; the first is associated with the dense scattering properties of tissues. Consequently, optical clearing (OC) methods have been developed to reduce tissue scattering by matching the tissue layers’ refractive indices via different protocols. The second factor is related to the illuminating wavefront and the size of the incident light beam. The present work monitored the optical transmittance of skeletal muscles after applying different OC approaches (physical OC using 99%-glycerol immersion and photothermal OC using IR-laser irradiation). First, the optical transmittance of the samples before and after the two OC procedures were compared, revealing a transmittance increase of 300% and 20%. Then, the laser beam wavefront aberrations were compensated in real-time by utilizing an active-adaptive Shack-Hartmann wavefront sensor system to provide an ideal illumination wavefront. Finally, the transmittance of the samples was compared using uncompensated and compensated laser wavefronts providing a 35% increase in the transmittance after aberrations compensation. Moreover, the aberration-free incident laser beam’s transmittance with different spot diameters was investigated. The results revealed that the larger beam diameter provided higher transmittance, hence higher optical penetration within the tissue.