UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS

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2022-07-01 17:51

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Conference: Bucharest University Faculty of Physics 2019 Meeting


Section: Biophysics; Medical Physics


Title:
Digital analysis of cytoskeleton actin fibers in images obtained by confocal fluorescence microscopy


Authors:
Călin Mircea RUSU (1)(2), Mihaela BACALUM (1), Antonia Teona DEFTU (2), Mihai RADU (1), Beatrice Mihaela RADU (2)


Affiliation:
1) Department of Life and Environmental Physics, “Horia Hulubei” National Institute of Physics and Nuclear Engineering, 30, Reactorului st, Măgurele, Romania

2) Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, 91–95, Splaiul Independenţei, Bucharest, Romania


E-mail
calin.rusu@nipne.ro


Keywords:
Cytoskeleton, actin, microscopy, confocal, digital, analysis


Abstract:
The cellular actin cytoskeleton is the main determinant of cell morphology and motility. The actin filaments network is important because a wide range of cellular processes can be monitored through the conformational changes occurring at its level. Classical methods, based on the measurement of the total fluorescence intensity in microscopy images, proved to be ineffective in identifying structural variations between different cell types or between cells of the same type undergoing different treatment conditions. The purpose of the study was to use a method of quantitative analysis based on identifying and segmenting cellular features in confocal fluorescence microscopy images. The FiberScore algorithm, a template-based segmentation method, is used for the effective detection and quantitative analysis of actin filaments. The detection of fibers is determined by the probability that a pixel line will belong to a fiber in the image. This probability is calculated by correlating pixel regions in the analyzed image with similar linear pixel structures under different orientations. Five types of cells were analyzed quantitatively: bEnd.3 (cerebral microvasculature endothelial cells), BJ (human skin fibroblasts), MG-63 (human tumor cells of osteosarcoma), OLN-93 (oligodendrocytes derived from primary brain cultures) and hiPSC (cardiomyocytes derived from pluripotent induced stem cells). They were fixed and stained with phalloidin-FITC for actin filaments. The analyzed parameters were: polarity, mean length, and number of actin filaments. Significant differences were found between the morphologies of the five cell lines. Also, there were differences between bEnd.3 cells undergoing treatment with various methylglyoxal concentrations. Based on our results, an extensive analysis of the conformational modifications of actin fibers can be performed to identify changes based on cellular morphology and/or applied pharmacological treatment. The algorithm allows the efficient separation between structures of interest and background noise, and it is a reliable analysis tool even for images of lower quality, due to its computational robustness.


References:

1) LICHTENSTEIN, N., B. GEIBER, Z. KAM, Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy, Cytometry A, 2003, 54A(1), 8–18.

2) PHAM, R.Q., Detection of Curvilinear Objects Using a Template Based Comparison: Applications to Microscopy and Solar Images, MSc, New Mexico State University, May 2013.

3) SUETSUGU, S., T. TAKENAWA, Regulation of cortical actin networks in cell migration, International Review of Cytology, 2003, 229, 245–286.