Final Report Summary - CARSFORSTEM (Probing the Fate of Stem Cells through the Development of a Multiplex Coherent Anti-Stokes Raman Scattering Imaging Technique)
Coherent anti-Stokes Raman scattering (CARS) microscopy has emerged in the past decade as a powerful multi-photon microscopy technique for rapid label-free imaging of organic materials and biological samples with sub-micrometer spatial resolution in three-dimensions and high chemical specificity. Among the various technical implementations of CARS microscopy reported to date, hyperspectral CARS imaging is receiving increasing attention due to its superior chemical specificity over single-frequency CARS. Analysing the resulting multidimensional data set in order to provide an efficient image visualization and a useful chemical interpretation is nontrivial. To overcome this complication, Dr Masia has developed a method of quantitative chemical imaging going from data acquisition using hyperspectral CARS to unsupervised analysis and visualization of the spatially resolved absolute concentrations of chemical components.
This approach can be extended easily to different hyperspectral imaging technique and can be used in a wide range of applications requiring fast and quantitative volumetric chemical imaging, from live cell microscopy to material science applications, providing an unsupervised technique to identify unknown chemical differences within and between samples.
The developed method has been proved to be an important tool to analyse CARS hyperspectral images to provide quantitative chemical information of the samples over a range of different investigations, including cell cycle studies, organoid composition, phase segregation in lipid membranes etc. In particular the method has been used by Dr. Masia and collaborators to study the uptake of saturated and unsaturated lipids by stem-cell derived adipocytes. The study has the potential to answer many open questions on the fundamental molecular mechanisms of lipid metabolism in cells which might be of key importance to tackle metabolic disease, such as obesity and diabetes, which greatly effect today’s societies.
The hyperspectral image analysis method has proven to be a key factor also for the study of CARS markers for the differentiation of mouse embryonic stem (mES) cells. Dr Masia has found that in mES cells undergoing differentiation into adipocytes CARS hyperspectral imaging reveals the presence of subcellular structures which are chemically different from the cytosol. After a comparison with fluorescent imaging, Dr Masia could conclude that such structures contains the fatty acid binding protein 4, which is thought to facilitate the transfer of fatty acids between extra- and intracellular membranes in adipocytes. This result represents a first step in the identification of the differentiation pathway undertaken by a stem cell before a change in morphology occurs using a label free microscopy technique, with important implications in the applicability of stem cell technology as a treatment for a series of diseases.
Dr Masia has furthermore developed a method to increase the acquisition rate by reducing the number of measured frequencies in the hyperspectral image, without losing significant spectral information. Dr. Masia has demonstrated that this method allows for an improvement in speed up to a factor of 30, enabling real-time chemical imaging and high-throughput high-content label-free microscopy.
This approach can be extended easily to different hyperspectral imaging technique and can be used in a wide range of applications requiring fast and quantitative volumetric chemical imaging, from live cell microscopy to material science applications, providing an unsupervised technique to identify unknown chemical differences within and between samples.
The developed method has been proved to be an important tool to analyse CARS hyperspectral images to provide quantitative chemical information of the samples over a range of different investigations, including cell cycle studies, organoid composition, phase segregation in lipid membranes etc. In particular the method has been used by Dr. Masia and collaborators to study the uptake of saturated and unsaturated lipids by stem-cell derived adipocytes. The study has the potential to answer many open questions on the fundamental molecular mechanisms of lipid metabolism in cells which might be of key importance to tackle metabolic disease, such as obesity and diabetes, which greatly effect today’s societies.
The hyperspectral image analysis method has proven to be a key factor also for the study of CARS markers for the differentiation of mouse embryonic stem (mES) cells. Dr Masia has found that in mES cells undergoing differentiation into adipocytes CARS hyperspectral imaging reveals the presence of subcellular structures which are chemically different from the cytosol. After a comparison with fluorescent imaging, Dr Masia could conclude that such structures contains the fatty acid binding protein 4, which is thought to facilitate the transfer of fatty acids between extra- and intracellular membranes in adipocytes. This result represents a first step in the identification of the differentiation pathway undertaken by a stem cell before a change in morphology occurs using a label free microscopy technique, with important implications in the applicability of stem cell technology as a treatment for a series of diseases.
Dr Masia has furthermore developed a method to increase the acquisition rate by reducing the number of measured frequencies in the hyperspectral image, without losing significant spectral information. Dr. Masia has demonstrated that this method allows for an improvement in speed up to a factor of 30, enabling real-time chemical imaging and high-throughput high-content label-free microscopy.