Abstract
Microscopy-based high-content and high-throughput analysis of cellular systems plays a central role in drug discovery. However, for contrast and specificity, the majority of assays require a fluorescent readout which always comes with the risk of alteration of the true biological conditions. In this work, we demonstrate a label-free imaging platform which combines chemically specific hyperspectral coherent anti-Stokes Raman scattering microscopy with sparse sampling and Bessel beam illumination. This enabled us to screen multiwell plates at high speed, while retaining the high-content chemical analysis of hyperspectral imaging. To demonstrate the practical applicability of the method we addressed a critical side effect in drug screens, namely, drug-induced lipid storage within hepatic tissue. We screened 15 combinations of drugs and neutral lipids added to human HepG2 liver cells and developed a high-content quantitative data analysis pipeline which extracted the spectra and spatial distributions of lipid and protein components. We then used their combination to train a support vector machine discriminative algorithm. Classification of the drug responses in terms of phospholipidosis versus steatosis was achieved in a completely label-free assay.
Highlights
For many years high-throughput high-content microscopy has played a central role in the discovery and development of therapeutics through the screening of phenotypes.[1]
Bessel beams have been recently proposed as a way to achieve rapid volumetric imaging via extended depth of field in nonlinear microscopy based on two-photon fluorescence,[20,21] second harmonic generation contrast,[22] and single-frequency stimulated Raman scattering.[23]
Their implementation for high-throughput hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy has not yet been shown. Their opposite use was discussed in ref.[24,25] to retrieve the axial structure of a laterally homogeneous sample from phase matching considerations and the directionality of the CARS emission
Summary
For many years high-throughput high-content microscopy has played a central role in the discovery and development of therapeutics through the screening of phenotypes.[1]. An example of a critical side effect that needs to be assessed in drug screens and is hard to detect in current fluorescent-based assays is drug-induced lipid storage within hepatic tissue. The onset of toxicity within hepatic tissue has led to the cancelation of clinical trials, and is the greatest cause of the withdrawal of commercially available therapeutics following approval.[4,5] Drug-induced liver injury (DILI) includes steatosis and phospholipidosis, which involve the production of cellular structures containing excess lipid. The wavelength shift is a direct signature of the frequency of the vibration which in turn depends on the type of chemical bond This scattering phenomenon usually produces a very weak signal, but can be strongly enhanced in CRS when two short laser pulses (denoted as pump and Stokes of optical frequencies ωp and ωs respectively) are used to coherently drive the vibrations at frequency ωp−ωs, such that Raman scattered light from coherently driven molecular vibrations in the focal volume constructively interferes
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