In vivo three dimensional cuticle intact Drosophila brain imaging using laser scanning optical resolution photoacoustic microscopy

Abstract

To study the structure and functions of the Drosophila brain, confocal microscopy is commonly used. However, surgical removal of the head cuticle of Drosophila is required because the cuticle hinders both the optical excitation and detection. Such invasive surgery may affect brain functions and prohibits long term monitoring. Targeting to the unmet need of surgery free procedure, here we propose laser scanning optical resolution photoacoustic microscopy (LSOR-PAM) for in vivo three dimensional cuticle intact Drosophila brain imaging. Cuticle intact Drosophila brains with cells in optic lobes expressing fluorescent protein DsRed, which serves as an optical absorber and thus a photoacoustic signal source, were imaged. Acquired in vivo 3D LSOR-PAM cuticle-intact brain images were cross-validated using their confocal microscopic counterparts with the cuticles being surgically removed. Acoustic and optical attenuation of the cuticles and degradation in spatial resolution caused by the cuticles were also measured, which explains the reason why LSOR-PAM outperforms confocal microscopy for cuticle intact brains. In addition, the optical absorption bleaching of DsRed expressing optic lobes as a function of the number of the repeated experiments was measured, verifying the LSOR-PAM long-term monitoring capability. In summary, we have demonstrated 3D LSOR-PAM of the Drosophila brain without invasive surgery for the first time. The focus of the future work will be on ways to explore its functional imaging capability on the cuticle intact Drosophila brain.