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Highly longitudinally polarized coherent anti-Stokes Raman scattering microscopy with improved spatial resolution
摘要： We proposed a method to enhance the longitudinally polarized component and improve the spatial resolution of radially polarized coherent anti-Stokes Raman scattering (CARS) microscopy by phase modulation. A specially designed phase pattern is applied onto the pump beam to suppress the radially polarized ?eld component at the focal region. With this modulation, the calculated intensity ratio between the longitudinally and radially polarized CARS ?eld is increased from 2.57 to 14.7, and the simulation of CARS imaging on a 120 nm polystyrene bead shows more than 3-fold spatial resolution improvement for both forward and backward detection. It is expected that this method could also be applied to other nonlinear optical imaging modalities for enhancing the longitudinally polarized component.
关键词： spatial resolution,microscopy,phase modulation,longitudinally polarized,coherent anti-Stokes Raman scattering
更新于2025-09-23 15:21:21
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High-throughput label-free molecular fingerprinting flow cytometry
摘要： Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm?1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.
关键词： high-throughput,astaxanthin,label-free,single-cell analysis,microfluidics,flow cytometry,Raman spectroscopy,Haematococcus lacustris,coherent anti-Stokes Raman scattering
更新于2025-09-19 17:15:36
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Reconstruction of coherent anti‐Stokes Raman scattering signals generated by means of laser pulses with asymmetric amplitude and phase
摘要： Time and frequency asymmetries in ultrashort chirped laser fields might appear as a consequence of dispersive propagation, pulse shaping techniques, or generation of auxiliary light pulses needed in nonlinear optics. Here, we try to find an answer to the question of how to solve analytically coherent anti‐Stokes Raman scattering (CARS) under asymmetric conditions of chirped femtosecond laser pulses. The approach breaks in two parts. One for the field amplitudes and the other for the phases. The former revolves around Gaussian dependences that, besides being rather common in ultrashort laser physics, can be arranged and mixed to reproduce spectrally asymmetric laser amplitudes. The latter is limited to field phases with cubic frequency dependence (i.e., second‐order chirp) whose asymmetry is simulated by adding a linear term to the quadratic phase. Both approximations for amplitudes and phases of the three laser pulses are mandatory to guarantee the solution to the complexity posed by the CARS problem. Comparisons with known experimental and numerical results support the validity of the model.
关键词： laser spectroscopy,coherent anti‐Stokes Raman scattering,ultrafast laser physics
更新于2025-09-11 14:15:04
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Fully convolutional networks in multimodal nonlinear microscopy images for automated detection of head and neck carcinoma: A pilot study
摘要： Background: A fully convolutional neural networks (FCN)-based automated image analysis algorithm to discriminate between head and neck cancer and non-cancerous epithelium based on nonlinear microscopic images was developed. Methods: Head and neck cancer sections were used for standard histopathology and co-registered with multimodal images from the same sections using the combination of coherent anti-Stokes Raman scattering, two-photon excited fluorescence, and second harmonic generation microscopy. The images analyzed with semantic segmentation using a FCN for four classes: cancer, normal epithelium, background, and other tissue types. Results: A total of 114 images of 12 patients were analyzed. Using a patch score aggregation, the average recognition rate and an overall recognition rate or the four classes were 88.9% and 86.7%, respectively. A total of 113 seconds were needed to process a whole-slice image in the dataset. Conclusion: Multimodal nonlinear microscopy in combination with automated image analysis using FCN seems to be a promising technique for objective differentiation between head and neck cancer and noncancerous epithelium.
关键词： digital pathology,semantic segmentation,diagnostics,second-harmonic generation,convolutional neural networks,two-photon excited fluorescence,spectral histopathology,image analysis,head and neck cancer,coherent anti-stokes Raman scattering
更新于2025-09-04 15:30:14
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Super-resolution microscopy and empirically validated autocorrelation image analysis discriminates microstructures of dairy derived gels
摘要： The food industry must capitalise on advancing technologies in order to optimise the potential from emerging ingredient technologies. These can aid in product optimisation and provide quantitative empirical data to which there is a fundamental physical understanding. Super-resolution microscopy provides a tool to characterise the microstructure of complex colloidal materials under near native conditions. Coherent Anti-Stokes Raman Scattering (CARS) microscopy was used to show the presence of fluorescent dye required for imaging does not affect gel microstructure and super-resolution Stimulated Emission Depletion (STED) microscopy is used to image four dairy derived gels. Image analysis has been developed based on 2D spatial autocorrelation, and a model that extracts parameters corresponding to a typical length of the protein domains and the inter pore distance. The model has been empirically validated through the use of generated images to show the fitting parameters relate to precise physical features. The fractal dimension is extracted from Fourier space analysis. The combination of STED microscopy and image analysis is sensitive enough to significantly differentiate samples based on whether gels were made from fresh or reconstituted milk, and whether gelation was induced through acidification or rennet addition. Rheometry shows that the samples exhibit different macroscopic behaviours, and these differences become increasingly significant with time. Samples can be differentiated earlier in the gelation process with imaging as compared to rheometry. This highlights the potential of STED imaging and image analysis to characterise the size of protein domains, pore spacing and the fractal dimensions of microstructures to aid product optimisation.
关键词： Stimulated Emission Depletion (STED) microscopy,Super-resolution microscopy,Fractal dimension,Coherent Anti-stokes Raman Scattering (CARS) microscopy,2D spatial autocorrelation analysis
更新于2025-09-04 15:30:14