Label-free Coherent Anti-Stokes Raman Scattering (CARS) imaging of the functional organisation of the cell nucleus

Session

LSA.1 - Label-free life science imaging

Authors

Peter Hoboth (1), Radek Machan (2), Dalibor Panek (2), Ales Benda (2), Pavel Hozak (1, 3, 4)

Affiliations

1. Department of Biology of the Cell Nucleus, Institute of Molecular Genetics, CAS v.v.i.
2. Imaging Methods Core Facility, BIOCEV, Faculty of Science, Charles University
3. Department of Epigenetics of the Cell Nucleus, Institute of Molecular Genetics, CAS v.v.i.
4. Microscopy Centre, Institute of Molecular Genetics, CAS v.v.i.

Keywords

Coherent Anti-Stokes Raman Scattering (CARS) microscopy, Nuclear lipids, Nuclear architecture

Abstract text

Coherent Anti-Stokes Raman Scattering (CARS) microscopy is label-free vibrational microscopy that allows visualization of  biomolecules in virtually non-treated biological samples. CARS microscopy is mostly used to visualize cellular lipids due to strong CARS signal of CH2 bonds frequently present in fatty acids. In the cells, lipids are present in the form of lipid droplets and lipid bilayers constitute cellular membranes. Lipids are also present in the nucleoplasm where they regulate various functions of the genome, such as gene expression. Nucleoplasmic lipid content is unevenly distributed and nucleoplasmic lipids are concentrated in the nucleoli, nuclear speckles and nuclear lipid islets. Here we used CARS microscopy to study the role of nuclear lipids in the functional organisation of the genome. We have detected heterogenous CARS signal in the cell nucleus and confirmed the specificity of the signal. We set on to identify the structural and functional components of the cell nucleus emitting CARS signal. Combination of the transmitted light and CARS microscopy revealed no correlation between CARS signal and nucleoli. Spectral imaging and linear unmixing revealed no correlation between CARS signal and fluorescently labeled speckles. Although CARS microscopy is often used to visualize cellular lipid content with no need for staining, CARS signal is emitted by CH2 bonds and therefore is not exclusively lipid specific. CH2 bonds are present also in proteins and nucleic acids. Therefore, we hypothesized that heterogeneity of nuclear CARS signal could represent different states of chromatin, e.g. heterochromatin vs. euchromatin. However, spectral imaging and linear unmixing revealed no correlation between CARS signal and DAPI stained heterochromatin. Our efforts to correlate nuclear CARS signal to specific nuclear structural and/or functional components continue. Those efforts will be beneficial for further application of label-free CARS microscopy to study the functional organization of the cell nucleus.