Coherent anti-Stokes Raman scattering (CARS) is a highly effective Non-Linear Optical (NLO) microscopy technique for label-free vibrational imaging of unperturbed samples. We present a novel scheme for video-rate wide-field CARS microscopy over a wide (up to 100-μm diameter) field of view, based on an Ytterbium amplified laser followed by white-light generation and optical parametric amplification. It records the full fingerprint region of the molecular vibrational spectrum and enables chemically-specific real-time characterization of fast dynamics with down to few-milliseconds time resolution. The system can perform either real-time imaging at single Raman shifts or acquire three-dimensional hypercubes, chemically richer in information.
Non-linear optical (NLO) microscopy techniques like coherent anti-Stokes Raman scattering (CARS) are highly effective tools for label-free vibrational imaging, allowing for chemical analysis of biological samples in their native state. We introduce video-rate wide-field CARS microscopy over a vast field of view (tens of micrometers) to enable real-time analysis of fast biological dynamics with down to few-millisecond time resolution. We generate stable broadband Stokes pulses in a YAG crystal using an amplified ytterbium laser source delivering 260-fs high-energy (μJ-level) pulses in the near-infrared. Our system combines fast and tunable single-wavelength real-time imaging with the acquisition of hypercubes, chemically more information-rich.
Recent oncology research highlights that senescence, once deemed beneficial in cancer treatments, can contribute to cancer relapse. Detecting therapy-induced senescent cells is challenging due to their complexity and lack of specific markers. Nonlinear optical (NLO) microscopy provides a fast, non-invasive, label-free detection solution. To distinguish between senescent and proliferating cells, here we present the development of a deep learning architecture based on multimodal NLO microscopy images coming from Stimulated Raman Scattering, Two Photon Excited Fluorescence and Optical Transmission. Despite limited labeled data, Transfer Learning, Data Augmentation, and Ensemble Learning techniques allowed us to achieve an accuracy over 90%. Ultimately, the predictions of the neural network are evaluated using the Grad-CAM visualization approach, which allows highlighting the most important features in the input images responsible for the labels assigned by the network. This work reveals the effectiveness of deep learning in senescence classification, potentially advancing treatment strategies.
Image classification using Deep Ensemble Learning and Transfer Learning methods is performed on a small, labeled dataset of multimodal nonlinear optical microscopy images coming from Stimulated Raman Scattering, Two Photon Excited Fluorescence and Optical Transmission, to differentiate proliferating cancer cells from senescent ones, a peculiar phenotype following an anti-cancer treatment responsible for tumour relapse. The superior performances of the Deep Ensemble Transfer Learning approach are compared with other less complex neural network architectures. Ultimately, the predictions of the neural network are evaluated using the Grad-CAM visualization approach, which allows highlighting the most important features in the input images responsible for the labels assigned by the network.
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