100-Second Large-Area Chemical Imaging by Integrated Four-Channel CARS Microscopy

Label-free coherent Raman scattering (CRS) microscopy enables chemical observation of intricate cellular and intercellular structures of biological tissues with submicron resolution and has become the most prominent stain-free alternative to gold-standard hematoxylin and eosin (H&E) histology. However, the imaging field of view (FOV) of current CRS microscopes is typically limited to less than a millimeter. Various attempts in instrumentation have been made to perform large-scale CRS mapping across an intact tissue, but the costly trade-offs are either imaging speed or spatial resolution. To address this challenge, we developed a four-channel coherent anti-Stokes Raman scattering (CARS) microscope, offering submicron imaging of 13 × 13 mm² large tissues within 100 s. Four miniaturized CARS systems, each equipped with a microelectromechanical system (MEMS) scanner, a micro-objective, and a collection fiber bundle, were fully integrated to perform ultrafast chemical mapping of intact unprocessed tissues with an electronically synchronized motorized stage. We demonstrated its capability by performing large-scale CARS imaging of mouse brain tissues and human glioma tissues. This label-free technique may pave the way for future clinical applications of stain-free pathology in the operating room.