Condensed Matter Physics Seminar

Prof. Wolfgang Langbein, Cardiff University, UK

Tuesday 27 May 2014, 1300-1400
C1 Physics Building

"Label-free quantitative chemical imaging with hyper-spectral CARS microscopy"

Coherent anti-Stokes Raman scattering (CARS) microscopy is a chemically-sensitive label-free multiphoton microscopy technique which has attracted much attention in the last decade for imaging in cell biology [1]. In CARS, the difference frequency of two optical fields coherently drives the vibrational resonances of molecules within the focal volume, and the light anti-Stokes scattered by the vibrations is detected.

We have developed a method to acquire and analyse hyperspectral CARS of organic materials and biological samples resulting in an unbiased quantitative chemical analysis [2]. The method employs singular value decomposition on the square root of the CARS intensity, providing an automatic determination of the components above noise. Complex CARS susceptibility spectra, which are linear in the chemical composition, are retrieved from the CARS intensity spectra using causality. We then use non-negative matrix factorization of the imaginary part and the non-resonant real part of the susceptibility together with a constraint on the total concentration to obtain susceptibility spectra of independently varying chemical components and their volume concentration. We call this method Factorization into Susceptibilities and Concentrations of Chemical Components (FSC3). The ability of the method to provide quantitative chemical analysis was shown on known lipid mixtures and by imaging lipid-rich mouse adipocytes. Cells were imaged in the CH-stretch vibrational range (2400-3800) cm-1 on a home built multimodal microscope [3]. We retrieve and visualize the most significant

chemical components with spectra given by water, lipid, and proteins, and we reveal the chemical structure of the cell with details visualized by the projection of the chemical contrast into a few relevant channels.

We furthermore show specificity to cellular compartments and components which traditionally need to be stained to be identified. Specifically, we imaged fixed U2OS cells with GFP tagged cyclin B1. The cells were arrested in different stages of their natural cycle.

In summary, FSC3 enables fast, quantitative and unsupervised volumetric chemical imaging, and should stimulate significant progress in the usefulness and applicability of CARS micro-spectroscopy to cell biology and material science.

[1] Andreas Zumbusch , Wolfgang Langbein, Paola Borri, Progress in Lipid Research 52, 615 (2013)

[2] Masia F, Glen A, Stephens P, Borri P, Langbein W, Anal. Chem. 85, 10820 (2013)

[3] Pope I, Langbein W, Watson P, Borri P, Opt. Express 21, 7096 (2013)