In this work, we have significantly enhanced the capabilities of the hyperspectral image analysis (HIA) first developed by Masia et al. 1 The HIA introduced a method to factorize the hyperspectral data into the product of component concentrations and spectra for quantitative analysis of the chemical composition of the sample. The enhancements shown here comprise (1) a spatial weighting to reduce the spatial variation of the spectral error, which improves the retrieval of the chemical components with significant local but small global concentrations; (2) a new selection criterion for the spectra used when applying sparse sampling2 to speed up sequential hyperspectral imaging; and (3) a filter for outliers in the data using singular value decomposition, suited e.g. to suppress motion artifacts. We demonstrate the enhancements on coherent anti‐Stokes Raman scattering, stimulated Raman scattering, and spontaneous Raman data. We provide the HIA software as executable for public use. © 2015 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons, Ltd.

In this work, we demonstrate quantitative volume determination of chemical components in three dimensions using hyperspectral coherent anti‐Stokes Raman scattering microscopy, phase‐corrected Kramers–Kronig retrieval of the coherent anti‐Stokes Raman scattering susceptibility and factorization into concentration of chemical components. We investigate the influence of the refractive index contrast between water and polymer beads (polystyrene and polymethylmethacrylate), showing that it leads mainly to concentration errors, while the spectral error is less affected. The volume of polystyrene beads of sizes from 200 nm to 3 μm is determined with 10% relative error and 1% absolute error in the region of interest. We furthermore establish the use of sodium chloride as non‐resonant reference material free of Raman‐active vibrational resonances. © 2016 The Authors Journal of Raman Spectroscopy Published by John Wiley & Sons Ltd.

Quantifying the chemical composition of unstained intact tissue and cellular samples with high spatiotemporal resolution in three dimensions would provide a step change in cell and tissue analytics critical to progress the field of cell biology. Label-free optical microscopy offers the required resolution and noninvasiveness, yet quantitative imaging with chemical specificity is a challenging endeavor. In this work, we show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy can be used to provide quantitative volumetric imaging of human osteosarcoma cells at various stages through cell division, a fundamental component of the cell cycle progress resulting in the segregation of cellular content to produce two progeny. We have developed and applied a quantitative data analysis method to produce volumetric threedimensional images of the chemical composition of the dividing cell in terms of water, proteins, DNAP (a mixture of proteins and DNA, similar to chromatin), and lipids. We then used these images to determine the dry masses of the corresponding organic components. The attribution of proteins and DNAP components was validated using specific well-characterized fluorescent probes, by comparison with correlative two-photon fluorescence microscopy of DNA and mitochondria. Furthermore, we map the same chemical components under perturbed conditions, employing a drug that interferes directly with cell division (Taxol), showing its influence on cell organization and the masses of proteins, DNAP, and lipids.

In this article, we report the number of cyclin B1 proteins tagged with enhanced green fluorescent protein (eGFP) in fixed U-2 OS cells across the cell cycle. We use a quantitative analysis of epifluorescence to determine the number of eGFP molecules in a nondestructive way, and integrated over the cell we find 10 4 to 10 5 molecules. Based on the measured number of eGFP tagged cyclin B1 proteins, knowledge of cyclin B1 dynamics through the cell cycle, and the cell morphology, we identify the stages of cells in the cell cycle.

We realized integrated photonics multi-waveguide devices for optical trapping and Raman spectroscopy of particles in a fluid. In these devices, multiple beams directed towards the device center lead to a local field enhancement around this center and thus counteract the effect of light concentration near the facets, which is a disadvantage of dual-waveguide traps. Thus, a trapping region is created around the center, where a single particle of a size in a wide range can be trapped and studied spectroscopically, free from the influence of surfaces. We report the design (including simulations), fabrication and performance demonstration for multi-waveguide devices, using our Si3N4 waveguiding platform as the basis. The designed ridge waveguides, optimized for trapping and Raman spectroscopy, emit narrow beams. Multiple waveguides arranged around the central microbath result from fanning out of a single input waveguide using Y-splitters. A second waveguiding layer is implemented for detection of light scattered by the trapped particle. For reliable filling of the device with sample fluid, microfluidic considerations lead to side channels of the microbath, to exploit capillary forces. The interference of the multiple beams produces an array of hot spots around the bath center, each forming a local trap. This property is clearly confirmed in the experiments and is registered in videos. We demonstrate the performance of a 2-waveguide and a 16-waveguide device, using 1 and 3 μm polystyrene beads. Study of the confined Brownian motion of the trapped beads yields experimental values of the normalized trap stiffness for the in-plane directions. The stiffness values for the 16-waveguide device are comparable to those of tightly focused Gaussian beam traps and are confirmed by our own simulations. The Raman spectra of the beads (in this work measured via an objective) show clear peaks that are characteristic of polystyrene. In the low-wavenumber range, the spectra have a background that most likely originates from the Si3N4 waveguides.