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Label-free volumetric quantitative imaging of the human somatic cell division by hyperspectral coherent anti-Stokes Raman scattering Arnica Karuna, Francesco Masia, Marie Wiltshire, Rachel J. Errington, Paola Borri, and Wolfgang Langbein Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04706 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019
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Analytical Chemistry
Label-Free Volumetric Quantitative Imaging of the Human Somatic Cell Division by Hyperspectral Coherent Anti-Stokes Raman Scattering Arnica Karuna,†,§ Francesco Masia,† Marie Wiltshire,‡ Rachel Errington,∗,‡ Paola Borri,¶ and Wolfgang Langbein∗,† †School of Physics and Astronomy, Cardiff University, The Parade, Cardiff CF24 3AA, UK ‡Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK ¶School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK §Current address: Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands E-mail:
[email protected];
[email protected] Abstract
is hindered by the complexity of the staining process and photobleaching. As alternative to fluorescence microscopy, label-free chemical sensitivity and high spatial resolution are provided by Raman scattering microscopy 5 , interrogating the vibrational resonances of endogenous molecules. However, spontaneous Raman scattering provides only weak signals, mandating long integration times and/or high excitation powers, limiting the applicability of the technique to imaging biological specimens. Specifically it is not suitable for live cells with cellular compartments that can move on a sub-second scale. Although recent developments toward light-sheet Fouriertransform Raman imaging 6 have the potential to improve the acquisition speed, Raman microscopy is also affected by autofluorescence background, which limits its present applicability to volumetric imaging and makes it incompatible with fluorescent labelling. Higher signals can be achieved in coherent Raman scattering (CRS), a non-linear optical technique in which molecular vibrations are driven coherently by a light intensity modulation, typically achieved by the interference of two laser beams of different wavelengths. The signal enhancement in CRS originates from the coherent superposition of the Raman scattered fields, with a factor given by the number of vibrational modes in the focal volume, which can reach 1010 for pure materials such as lipids 7 . CRS can be measured as coherent anti-Stokes Raman scattering (CARS) 8 , or stimulated Raman scattering (SRS) 9 , both having their advantages and drawbacks 7,10–12 . CARS is generally easier to implement experimentally, since it occurs at a different wavelength compared to that of the exciting beams, thus the signal can be easily separated via an optical bandpass filter, and is blue shifted compared to the one-photon excited fluorescence background. On the other hand, it requires a more in depth data analysis, due to the contribution of vibrationally non-resonant backgrounds. Specifically, the CARS intensity can be described as IC ∝ |χ|2 = |χR +χNR |2 , where χR (χNR ) is the vibrationally resonant (non-resonant) component of the third-order susceptibility corresponding to the CARS process. χR has both real and imaginary parts, while χNR is due to an electronic contribution, and is real for materials without two-photon absorption, which is the case for water, proteins, DNA, and lipids, for excitation pulses with wavelengths in the biological window 7 , 700 - 1300 nm. Therefore, one can rewrite IC ∝ |χR |2 +2
