Label-Free Volumetric Quantitative Imaging of the Human Somatic Cell Division by Hyperspectral Coherent Anti-Stokes Raman Scattering

Abstract

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.

Label-free volumetric quantitative imaging of human osteosarcoma cells by hyperspectral coherent anti-Stokes Raman scattering

“…In addition to the analysis presented here, CARS microscopy has also been applied to the study of mitosis and the effects of drug treatment in U-2OS cells . 17 The data shown in this paper provides additional information about interphase, the stage in which the cells spend most of their time during the cell cycle.…”

“…Notably, lipids are detected as a well-defined component in these samples due to the high abundance of C-H bonds in lipids. The components with small (χ), such as DNAP, are harder to separate , 17 and appear as mixed proteins and DNA in varying fractions. Regardless of this, we believe these results are impressive due to the label-free determination of the dry masses of not only lipids, but also proteins and DNAP in non-lipid rich cells containing mostly water.…”

“…The cells were cultured as described in Ref. 17, and seeded on #1 coverslips placed into a single well of a multi-well plate. They were allowed to adhere and proliferate for 24 , followed by (χ) and FSC 3 components W, 1, 2, and 3 corresponding to sum water, protein, DNAP, and lipid respectively.…”

“…Assuming that for IFDs above 3100 cm −1 , (χ) is dominated by the water component, while the organic components have significant (χ) in the range (2800-3100) cm −1 , we follow the method described in Ref. 17. Briefly, we subtract a fraction α of the water spectrum (χ W ) from the component spectra, resulting in the dry spectrum (χ dry ) = (χ)−α (χ W ).…”

“…Recently, we reported a quantitative study of mitosis in human cells using chemically specific, label-free hyperspectral CARS microscopy . 17 Briefly, the dry masses of various biomolecules, namely, proteins, a mixture of DNA and proteins, which we call here DNAP, and lipids were calculated over the whole cell and in the chromosomal regions. The attribution of the FSC 3 components to various cell compartments/biomolecules was validated using fluorescence labelling of the cells with well-characterized fluorophores, DAPI and Mitotracker-orange.…”