The third-order nonlinear nature of Broadband CARS means it has the inherent capability to produce label-free images using vibrational information as contrast. The speed of the technique is several orders of magnitude greater than spontaneous Raman spectroscopy. This has implications for enabling diagnostics in areas such as histology, immunology and cytology. The major drawback in BCARS currently preventing these applications is mainly the nonresonant background, present due to degenerate four-wave mixing. This background can be removed using the tensorial properties of the electronic susceptibility. This technique is known as spectral interferometric polarization CARS (SIP-CARS). We show an implementation of SIP-CARS on highly resonant polymer beads for spectroscopic imaging using two sequential BCARS scans to probe different components of the susceptibility tensor.
The application of Broadband CARS to cell imaging studies has thus far been limited to those where high contrast features are present, such as lipids and exogenously introduced tags. This is due to the inherent low SNR obtained in BCARS from the low density of oscillators in single cells coupled with the non-resonant background present in all media which distorts the measured signal. In this paper, we show that an autoencoder which we named VECTOR2, trained on simulated spectra, can accurately perform NRB removal of recorded BCARS images of unstained biological specimen. This allows cell imaging comparable in time to spontaneous Raman imaging with high bandwidth and resolution. The introduction of standard baseline flattening prior to NRB removal preserves the image structure while removing artefacts from raster scanning and optical noise. This results in a hyperspectral image of the NRB-free BCARS signal which is linear in the sample concentration and has a spectrum that is very similar to the spontaneous Raman spectrum.
MicroRNAs are small ~22 nucleotide RNA sequences that are guided to the 3’ untranslated region (UTR) of protein-coding target mRNA sequences. One particular microRNA, miR155, plays a remarkable role in the immune system, where it is essential for mounting appropriate immune responses. However, its dysregulation has been identified in multiple inflammatory disorders such as Multiple Sclerosis (MS), arthritis, psoriasis and colitis. More specifically, miR-155 has been found to be elevated in the serum and brain lesions of MS patients. Importantly, therapeutic inhibition of miR-155 can inhibit progression of the MS disease model. One of us has identified that macrophages are major contributor to miR-155 elevation in the MS disease model, whilst its inhibition specifically in macrophages can limit the disease. Here macrophages were isolated from the femur and tibia of wild-type (WT) mice and mice with a knock-out (KO) of the gene regulating miR-155 production, and were cultured in-vitro and stimulated with lipopolysaccharide (LPS) to simulate an immune response. Cells were then prepared for spectral analysis by FTIR imaging with a Perkin-Elmer Spotlight 400 imaging microscope. After pre-processing the dimensionality of spectra were reduced using principal components analysis, kernel-PCA and universal manifold application and projection (UMAP) and classified using a support vector machine algorithm, delivering a classification performance approaching F1~0.89. Spectral features differentiating WT and KO classes were observed across the fingerprint region with no single spectral marker being the sole source of differentiation of the downstream molecular events. This study exemplifies the challenge in spectral discrimination of the complexity of molecular events in ex-vivo models of immune dysregulation.
Multi-modal spectroscopic analysis of biological systems may offer an improved overall non-invasive biophotonic metric of the status of the system, further enhancing the diagnostic and prognostic capabilities of these technologies. In the present study macrophages were extracted from wild-type mice and mice with a knock-out of the gene regulating miR-155, which has been observed to occur in patients with various autoimmune disorders, including multiple sclerosis (MS). Macrophages were stimulated in-vitro to produce an immune response and were then screened spectroscopically with FTIR and Raman spectroscopy (at 532nm and 660nm). Low, medium and high level data fusion strategies for classification of response to stimulation and miRNA regulation were piloted, using downstream principal components analysis-support vector machine classifiers to test the impact of these strategies on classification performance. These techniques allowed the development of a combined highlevel data-fusion, classification pipeline with a high level of classification accuracy (F1<0.9), with reduced variability in performance. Our proposed spectroscopic assay-data fusion strategy may provide an adjunct to clinical screening and diagnosis of various autoimmune disorders whose aetiology is associated with genetic dysregulation.
Raman micro-spectroscopy (RMS) is a powerful technique for the identification, classification, and diagnosis of cancer cells and tissues.1 The requirement for long acquisition times of 1-30 s have impeded clinical application. The slow acquisition time can be overcome by the use of coherent Raman scattering (CRS), a class of thirdorder nonlinear optical spectroscopies that employ a sequence of light pulses to set-up a vibrational coherence within the ensemble of molecules inside the laser focus. The two most widely employed CRS techniques are coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) both of which achieve extremely high acquisition speeds up to the video rate, but traditional architectures are limited in terms of “single frequency” detection resulting from the use of picosecond pump and Stokes pulses with an optical bandwidth of a few wavenumbers. An important breakthrough has been recently achieved by the Cicerone group2 using a femtosecond Er:fiber laser oscillator followed by two erbium doped fiber amplifier arms; one arm is frequency doubled to generate narrow-band pulses at 770 nm with a flat-top 3.8 ps temporal profile, while the other is spectrally broadened in a highly non-linear fiber to generate a broad supercontinuum spanning the 900–1350 nm wavelength region. This pulse combination enables extraction of the CARS response by both the two-color and the three-color mechanism. While all previous broadband CARS systems failed to provide a low-noise spectrum in the fingerprint region, this approach has enabled Raman spectra in the whole biologically relevant frequency region (500–3500 cm-1) to be captured with 10 cm-1 resolution and 3.5 ms acquisition. Here, we provide guidance on the initial setup and optimization of this bCARS micro-spectroscopy system, with specific examples of the common pitfalls encountered during the setup. This is particularly useful for those coming from a background of designing spontaneous Raman spectroscopy systems for biomedical applications.
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