Teitl: Applications of coherent anti-Stokes Raman scattering (CARS) microscopy to cell biology

Cardiff University

Prif Ymchwiliwr

Manylion y Prosiect
Dyddiad dechrau: 02.07.2012
Dyddiad gorffen: 05.05.2015

CARS microspectroscopy with a suited subsequent analysis as used in this work (phase corrected Kramers-Kronig (PCKK) and factorization into concentrations of chemical components (FSC3), see section 3.3) provides chemically specific and quantitative information about the materials under study with three-dimensional spatial
resolution in the sub-micrometer range. To verify the volumetric accuracy of the method, polymer beads of known sizes were imaged and their volumes determined from the FSC3 components were found to be in close agreement with their actual volumes (section 3.4.2). The imaging optics and the refractive index of the samples
were found to have no bearing on the accuracy of this quantitative analysis. This project focussed on studying cell division which is responsible for the evolution of life from its unicellular origins to multicellularity. However, if cell division is not regulated, it can lead to diseases like cancer. This necessitates studies on cell division
to reveal clues pertinent to the nature of the cancer itself, which can aid in the development of cancer treatments. In this project, CARS microscopy was used to study fixed U-2OS (ATCC HTB-96) osteosarcoma cells transfected with a G2M Cell Cycle Phase Marker (GE Healthcare, UK), eGFP to label cyclin B1 which is
expressed during the mitotic phase of the cell cycle.
Starting with feasibility studies to demonstrate the usability of CARS in detecting the chemical contrast between different cell components i.e., chromatin, protein and lipid, the origins of various FSC3 components was established using correlative TPF. While the chromatin component is certainly nuclear in its origin, the components corresponding to protein and lipids are not colocalized to any one organelle. The suitability of CARS to image the cells in a quantitative way has been demonstrated through volumetric estimations of the relevant FSC3 components, determining the dry masses of the lipid, protein and chromatin components. Furthermore, the effects of two types of anti-cancer drugs on the cells have been studied. We found that although Taxol produces no detectable differences in the cell’s CARS component spectra, the changes in the morphology and intracellular distribution of chromatin induced due to Taxol treatment, are discernible. In this way, we have probed the indirect effect of Taxol on microtubules which play an important role in the movement/distribution of chromosomes during mitosis. This is a new way of studying the action of this drug on cells, without the need for introducing exogenous labels. Along with Taxol, another drug, ICRF-193, a  topoisomerase II poison forms an important part of chemotherapy against osteosarcoma. The effects of both
these drugs on cellular function have been documented in literature as discussed in chapter 4. In this project, we discovered, in the first CARS based study of its kind, that ICRF-193 changes the FSC3 component localized within the nucleus both spectrally and in its spatial distribution. In these cells, the distribution of the FSC3 component corresponding to chromatin is confined/more pronounced in the intranuclear punctate regions of the control sample (see Fig. 4.31) while this component is homogeneously distributed over the nuclei in drug treated cells. Additionally, on analyzing the control sample not aiming to find a common spectral bases for the
drug treated and the control cells, we observe in addition to the nuclear component corresponding to the punctates, another nuclear component similar in its spectrum and spatial distribution to the nuclear component in non-drug treated cells. This is an important result, which if proven on statistically significant populations of cells and on live cells, opens up an interesting application of CARS microscopy to study drug treated populations and their phenotype post treatment. Going from single cells to interacting cell assemblies, 3D models of organs, organoids grown in lab were also imaged using CARS and a similar chemical specificity was
observed for these samples as for the cells, revealing their three-dimensional organization and chemical composition in a label-free way.

Diweddarwyd y tro diwethaf ar 2020-05-05 am 15:00