Unified Vibrational and Multiphoton Label-Free Nonlinear Microscopy for Simultaneous Chemical and Structural Imaging

Nonlinear microscopy enables label-free imaging by deriving contrast from the intrinsic spectroscopic responses of specimens, thereby offering a valuable tool for biomedical applications. Often, clinical imaging systems implement either multiphoton or vibrational contrast, not both. Consequently, most clinically deployed label-free nonlinear microscopes lack a robust, complementary contrast palette suitable for investigating the morphofunctional features of heterogeneous specimens. This deficiency limits not only the analytical capabilities of the nonlinear microscope but also its diagnostic utility. A main reason for this disregard is that the various imaging modalities impose distinct and stringent requirements on the excitation source and the detection chain. In this contribution, we propose a strategy that targets both multiphoton and vibrational contrasts to achieve a robust, complementary contrast palette. The approach emerges from a systematic investigation of readout schemes and provides engineering criteria to tailor the detection chain and thus maximize quantitative performance. In concert with this detection strategy, we present a compact laser source that drives vibrational coherences while simultaneously exciting multiphoton signals. We validate the resulting imaging platform using two rodent case studies: one involving a naturally occurring metastatic cancer in a mouse and another relying on an allogeneic mammary cancer model in a rat. Owing to its dimensions, cost, and versatility, we anticipate that this biophotonics tool will readily find its way into clinical applications.