Confocal Raman Microscopy with Adaptive Optics

Abstract: Confocal Raman microscopy, a highly specific and label-free technique for the microscale study of thick samples, often presents difficulties due to weak Raman signals. Inhomogeneous samples introduce wavefront aberrations that further reduce these signals, requiring even longer acquisition times. In this study, we introduce adaptive optics to confocal Raman microscopy for the first time to counteract such aberrations, significantly increasing the Raman signal and image quality. The method is designed to integrate seamlessly with existing commercial microscopes without hardware modifications. It uses a wavefront sensorless approach to derive aberrations using an optofluidic, transmissive spatial light modulator that can be attached to the microscope nosepiece. Our experimental results demonstrate the compensation of aberrations caused by artificial scatterers and mouse brain tissue, improving spatial resolution and achieving up to 3.5-fold signal enhancements. Our results provide a basis for the molecular label-free study of biological systems at greater imaging depths.