Confocal Raman Microscopy with Adaptive Optics
Confocal Raman microscopy is a precise and label-free technique for analyzing thick samples at the microscale, but its use is often limited by weak Raman signals. Sample inhomogeneities introduce wavefront aberrations, further diminishing signal strength and requiring longer acquisition times. In this study, we present the first application of Adaptive Optics in confocal Raman microscopy to correct these aberrations, achieving substantial improvements in signal intensity and image quality. This approach integrates seamlessly with commercial microscopes without the need for hardware modifications. It utilizes a wavefront sensorless method, relying on an optofluidic, transmissive spatial light modulator attached to the microscope nosepiece to measure and correct aberrations. Experimental validation shows effective correction of aberrations in artificial scatterers and mouse brain tissue, enhancing spatial resolution and increasing signal intensity by up to 3.5 times. These results establish a foundation for deeper, label-free molecular imaging of biological systems.
Results
We introduce the first application of Adaptive Optics (AO) in confocal Raman microscopy (CRM) to correct sample-induced aberrations, improving signal intensity, contrast, and spatial resolution. This innovation achieves up to a 3.5-fold signal enhancement, significantly reducing acquisition times while maintaining signal-to-noise ratios.
Our approach employs a transmissive wavefront shaper mounted on the microscope nosepiece, utilizing a wavefront sensorless method to retrieve aberration data. By defining "spectral guide stars" using Raman spectral information, we ensure a monotonic relationship between aberration magnitude and the optimization metric, crucial for hill-climbing routines. These guide stars are effective even with weaker axial sectioning systems like line-scanning Raman setups.
We demonstrate correction of spherical aberrations from refractive index mismatches and nonspherical aberrations in mouse brain tissue. While our process is not yet optimized for speed, reducing the number of measurements (e.g., from 300 to 21) could improve efficiency by over tenfold.
Our setup corrects both excitation and emission paths using a deformable phase plate (DPP), compatible with existing commercial microscopes. Future refinements, including better metrics and faster sensing techniques, could further enhance AO’s performance, paving the way for deeper, label-free imaging in biological studies.
Comments
Post a Comment