multiplex-chemical-imaging-based-on-broadband-stimulated-raman

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

a-multimodal-imaging-framework-to-advance-phenotyping-living-label

A Multimodal Imaging Framework to Advance Phenotyping of Living Label-free Breast Cancer Cells

We present multimodal confocal Raman micro-spectroscopy (RS) and tomographic phase microscopy (TPM) for quick morpho-chemical phenotyping of human breast ...

Politecnico di Milano

Massachusetts Institute of Technology

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques.

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells.

Accurate, rapid and non-invasive cancer cell phenotyping is a pressing concern across the life sciences, as standard immuno-chemical imaging and omics require extended sample manipulation. Here we combine Raman micro-spectroscopy and phase tomography to achieve label-free morpho-molecular profiling of human colon cancer cells, following the adenoma, carcinoma, and metastasis disease progression, in living and unperturbed conditions. We describe how to decode and interpret quantitative chemical and co-registered morphological cell traits from Raman fingerprint spectra and refractive index tomograms. Our multimodal imaging strategy rapidly distinguishes cancer phenotypes, limiting observations to a low number of pristine cells in culture. This synergistic dataset allows us to study independent or correlated information in spectral and tomographic maps, and how it benefits cell type inference. This method is a valuable asset in biomedical research, particularly when biological material is in short supply, and it holds the potential for non-invasive monitoring of cancer progression in living organisms.

ArticleTotal : 2	Year
Publication title Cited by 8	2022
Publication title	2025

Article	Year
Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques. International journal of molecular sciences\| PubMed ID: 33187392
Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells. Biosensors\| PubMed ID: 37998148	2023
Label-free morpho-molecular phenotyping of living cancer cells by combined Raman spectroscopy and phase tomography. Communications biology\| PubMed ID: 38951178	2024

Arianna Bresci

Biography

JoVE Journal Publications

Other Publications