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.
Recently, anticancer treatments were discovered to induce cell senescence other than death, a critical phenotype driving tumor recurrence. This calls for the development of safe, precise, and rapid tools to reveal critical therapy-induced senescence (TIS). Here, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful technique to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these NLO signals from label-free TIS cells and controls, we unveiled quantitative all-optical traits of early-stage TIS, monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an shrinking of mitochondrial networks. CARS and SRS revealed lipid vesicles accumulation in cytoplasms. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We believe our results will strongly influence anticancer pre-clinical studies and translated clinical applications, constituting a quick, non-invasive, and accurate aid to expose TIS manifestation in tumors.
Coherent anti-Stokes Raman Scattering (CARS) microscopy is a label-free vibrational imaging technique that delivers chemical maps of cells and tissues. CARS employs two narrowband picosecond pulses (pump and Stokes) that are spatiotemporally superimposed at the sample plane to probe a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite many technological advancements, BCARS microscopes still struggle to image biological samples spanning the entire Raman active region of biological samples (400-3100 cm-1). Here, we demonstrate a novel BCARS method to answer this need. Our experimental setup is based on a femtosecond fiber laser at 1035 nm and 2 MHz repetition rate, thus delivering high energy pulses used for generating sub-20 fs broadband Stokes pulses by white-light continuum in a bulk YAG crystal, a compact and alignment-insensitive technique. Combining them with narrowband picosecond pulses, we can generate a CARS signal with high (< 10 cm-1) spectral resolution in the entire Raman window exploiting both two-color and three-color excitation mechanisms. The system is equipped with a home-made transmission microscope to image cells and tissue at high-speed (< 3 ms) and large field of views. Using a post-processing pipeline, we deliver high-quality chemical maps, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, unveiling the path for applications in histopathological settings.
Recent studies have shown that common anticancer treatments can induce cell senescence rather than death, a critical phenotype governing tumor recurrence. This calls for the urgent development of safe, precise, and quick tools to unveil critical Therapy-Induced Senescence (TIS). Merging different coherent Raman and multiphoton techniques, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful tool to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these diverse techniques applied on label-free TIS cells and controls, we exposed quantitative hallmarks of early TIS, confirmed by comparing different optical signals monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an early shrinking of mitochondria. CARS and SRS revealed lipid vesicles overproduction and accumulation. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We consider our results will strongly influence anticancer pre-clinical studies and translated clinical applications, helping to identify quickly, non-invasively, and quantitatively TIS in human tumors.
Cancer research recently revealed that anticancer therapies can cause cell senescence instead of death, a phenotype governing tumor relapse. Developing safe, quick, and precise tools to spot such therapy-induced senescence (TIS) is an urgency.
We present multimodal coherent Raman and multiphoton nonlinear optical microscopy as powerful to unveil TIS, via a home-built microscope including forward-detected Stimulated Raman Scattering, forward and epi-detected Coherent Anti-Stokes Raman Scattering, Two-Photon Excited Fluorescence and Second-Harmonic Generation modalities. We exposed early TIS in human cancer cells, confirmed comparing diverse signals during therapy period.
We consider our findings will strongly influence anticancer practices, helping prevent tumor recurrence.
Coherent Raman scattering (CRS) microscopy is a powerful third-order nonlinear optical technique for label-free chemical identification of molecules based on their intrinsic vibrational spectrum. It can reach video-rate imaging speed and detect very dilute species. I will review the working principles and state of the art of this technique, presenting our recent results and perspectives. I will also show how artificial intelligence methods can be employed effectively to increase the signal-to-noise ratio and extract the relevant information from the large experimental hyperspectral datacubes.
Coherent anti-Stokes Raman scattering is an extremely powerful non-linear optical (NLO) microscopy technique for label-free vibrational imaging allowing for chemical characterization of biological samples in their native state. We introduce here video-rate wide-field signal generation and acquisition over a large field of view (tens of micrometers) allowing for real-time investigation of fast biological dynamics. To this aim, our innovative approach employs an amplified femtosecond ytterbium laser source delivering high energy (≈ µJ) pulses in the near-infrared at 1035-nm central wavelength and 2-MHz repetition rate from which pump and Stokes beams are generated. Narrowband pump pulses with ≈1.1 nm bandwidth (10 cm−1) guarantee sufficient spectral resolution for the vibrational Lorentian peaks. Broadband Stokes pulses in the 1100 to 1500 nm range are produced via supercontinuum generation in 10-mm YAG crystal and amplified in a non-collinear optical parametric amplifier (NOPA). This allows us to acquire hypercubes that cover the entire fingerprint region of the molecular vibrational spectrum, the richest in chemical information. Our results pave the way for future clinical diagnostics applications with video-rate imaging capabilities.
We propose a novel approach to broadband coherent anti-Stokes Raman scattering (B-CARS) based on a femtosecond laser at 1035 nm and 2 MHz repetition rate. These features of the driving laser enable white-light continuum generation in bulk media, employed as broadband Stokes. In this way, we demonstrate state-of-the-art acquisition speed (<1 ms/pixel) with unprecedented sensitivity (≈14.1 mmol/L) when detecting dimethyl-sulfoxide in water, covering the whole fingerprint region. To further enhance the performance of the system, we designed an innovative spectral denoiser based on a convolutional neural network, coupled with a post-processing pipeline to distinguish different chemical species of biological tissues.
We present a novel broadband coherent anti-Stokes Raman scattering (B-CARS) approach, employing a femtosecond fiber laser at 1035 nm and 2 MHz repetition rate. These characteristics of the driving laser enable to generate broadband Stokes pulses through white-light supercontinuum in bulk media and to use higher pulse energy to shine the sample, thus producing stronger signals. In this way, we demonstrate state-of-the-art acquisition speed (<1 ms/pixel) with unprecedented sensitivity (≈14.1 mmol/L), covering the whole fingerprint vibrational region. The B-CARS microscope, coupled with a deep learning-based post-processing pipeline, delivers high-quality hyperspectral images identifying the main Raman features of heterogeneous biological tissues.
We present several approaches to high-speed broadband stimulated Raman scattering (SRS) and broadband coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy for label-free chemical identification of molecules through their intrinsic vibrational spectrum. (1) A 32-channels lock-in amplifier for parallel detection of broadband SRS spectra. (2) A broadband CARS microscope covering the entire fingerprint with down to 1-ms pixel dwell time, based on a low-repetition-rate amplified laser allowing white-light supercontinuum generation in bulk media. (3) Artificial-intelligence-based methods to enhance the signal-to-noise ratio and remove the non-resonant background signal from CARS spectra. (4) Video-rate wide-field CARS microscopy over the fingerprint region.
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