Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Nov;87(11):2757-2773.
doi: 10.1002/jemt.24648. Epub 2024 Jul 10.

Label-free three-dimensional imaging and quantitative analysis of living fibroblasts and myofibroblasts by holotomographic microscopy

Francesca Sbrana  1 , Flaminia Chellini  2 , Alessia Tani  2 , Martina Parigi  2 , Rachele Garella  3 , Francesco Palmieri  3 , Sandra Zecchi-Orlandini  2 , Roberta Squecco  3 , Chiara Sassoli  2
Affiliations

Label-free three-dimensional imaging and quantitative analysis of living fibroblasts and myofibroblasts by holotomographic microscopy

Francesca Sbrana et al. Microsc Res Tech. 2024 Nov.

Abstract

Holotomography (HT) is a cutting-edge fast live-cell quantitative label-free imaging technique. Based on the principle of quantitative phase imaging, it combines holography and tomography to record a three-dimensional map of the refractive index, used as intrinsic optical and quantitative imaging contrast parameter of biological samples, at a sub-micrometer spatial resolution. In this study HT has been employed for the first time to analyze the changes of fibroblasts differentiating towards myofibroblasts - recognized as the main cell player of fibrosis - when cultured in vitro with the pro-fibrotic factor, namely transforming growth factor-β1. In parallel, F-actin, vinculin, α-smooth muscle actin, phospho-myosin light chain 2, type-1 collagen, peroxisome proliferator-activated receptor-gamma coactivator-1α expression and mitochondria were evaluated by confocal laser scanning microscopy. Plasmamembrane passive properties and transient receptor potential canonical channels' currents were also recorded by whole-cell patch-clamp. The fluorescence images and electrophysiological results have been compared to the data obtained by HT and their congruence has been discussed. HT turned out to be a valid approach to morphologically distinguish fibroblasts from well differentiated myofibroblasts while obtaining objective measures concerning volume, surface area, projection area, surface index and dry mass (i.e., the mass of the non-aqueous content inside the cell including proteins and subcellular organelles) of the entire cell, nuclei and nucleoli with the major advantage to monitor outer and inner features in living cells in a non-invasive, rapid and label-free approach. HT might open up new research opportunities in the field of fibrotic diseases. RESEARCH HIGHLIGHTS: Holotomography (HT) is a label-free laser interferometric imaging technology exploiting the intrinsic optical property of cells namely refractive index (RI) to enable a direct imaging and analysis of whole cells or intracellular organelles. HT turned out a valid approach to distinguish morphological features of living unlabeled fibroblasts from differentiated myofibroblasts. HT provided quantitative information concerning volume, surface area, projection area, surface index and dry mass of the entire fibroblasts/myofibroblasts, nuclei and nucleoli.

Keywords: fibrosis; holotomographic microscopy; label‐free; myofibroblasts; quantitative phase imaging.

PubMed Disclaimer

References

REFERENCES

    1. Adam, S. A. (2017). The nucleoskeleton. Cold Spring Harbor Perspectives in Biology, 9(2), a023556. https://doi.org/10.1101/cshperspect.a023556
    1. Barer, R. (1953). Determination of dry mass, thickness, solid and water concentration in living cells. Nature, 172(4389), 1097–1098. https://doi.org/10.1038/1721097a0
    1. Cappitti, A., Palmieri, F., Garella, R., Tani, A., Chellini, F., Salzano De Luna, M., Parmeggiani, C., Squecco, R., Martella, D., & Sassoli, C. (2023). Development of accessible platforms to promote myofibroblast differentiation by playing on hydrogel scaffold composition. Biomaterials Advances, 155, 213674. https://doi.org/10.1016/j.bioadv.2023.213674
    1. Chellini, F., Tani, A., Vallone, L., Nosi, D., Pavan, P., Bambi, F., & Sassoli, C. (2018). Platelet‐rich plasma prevents in vitro transforming growth factor‐β1‐induced fibroblast to myofibroblast transition: Involvement of vascular endothelial growth factor (VEGF)‐A/VEGF receptor‐1‐mediated signaling. Cells, 7(9), 142. https://doi.org/10.3390/cells7090142
    1. Chellini, F., Tani, A., Vallone, L., Nosi, D., Pavan, P., Bambi, F., Zecchi‐Orlandini, S., & Sassoli, C. (2018). Platelet‐rich plasma and bone marrow‐derived mesenchymal stromal cells prevent TGF‐β1‐induced myofibroblast generation but are not synergistic when combined: Morphological in vitro analysis. Cells, Tissues, Organs, 206(6), 283–295. https://doi.org/10.1159/000501499

LinkOut - more resources