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Tracking of fluorescence nanoparticles with nanometre resolution in a biological system: assessing local viscosity and microrheology

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Abstract

The aim of the study was to establish a user-friendly approach for single fluorescence particle 3D localization and tracking with nanometre precision in a standard fluorescence microscope using a point spread function (PSF) approach, and to evaluate validity and precision for different analysis methods and optical conditions with particular application to microcirculatory flow dynamics and cell biology. Images of fluorescent particles were obtained with a standard fluorescence microscope equipped with a piezo positioner for the objective. Whole pattern (WP) comparison with a PSF recorded for the specific set-up and measurement of the outermost ring radius (ORR) were used for analysis. Images of fluorescent particles were recorded over a large range (about \(7\,\upmu \text{ m }\)) of vertical positions, with and without distortion by overlapping particles as well as in the presence of cultured endothelial cells. For a vertical range of \(6.5\,\upmu \text{ m }\), the standard deviation (SD) from the predicted value, indicating validity, was 9.3/8.7 nm (WP/ORR) in the vertical and 8.2/11.7 nm in the horizontal direction. The precision, determined by repeated measurements, was 5.1/3.8 nm in the vertical and 2.9/3.7 nm in the horizontal direction. WP was more robust with respect to underexposure or overlapping images. On the surface of cultured endothelial cells, a layer with 2.5 times increased viscosity and a thickness of about \(0.8\,\upmu \text{ m }\) was detected. With a validity in the range of 10 nm and a precision down to about 3–5 nm obtained by standard fluorescent microscopy, the PSF approach offers a valuable tool for a variety of experimental investigations of particle localizations, including the assessment of endothelial cell microenvironment.

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Acknowledgments

We are grateful to Cathrin Dressler for providing for us the fluorescent nanoparticles, to Thomas Meinel for his help in the data analysis and to Klaus Affeld for his help in capillary viscometer measurements. The work of Alex Marki was supported by the Ernst von Leyden Stipend of the Berlin Cancer Society (Berliner Krebsgesellschaft e.V., Berlin, Germany). The project was supported by the Schüchtermann-Foundation (Germany, Dortmund), and the support by H. Warnecke is acknowledged.

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Authors and Affiliations

  1. Department of Physiology, Charité Universitätsmedizin, Charitéplatz 1, 10117 , Berlin, Germany

    Alex Marki, Eugeny Ermilov, Andreas Zakrzewicz & Axel R. Pries

  2. Department of Gerontology and Pathophysiology, University of Pécs, Pécs, Hungary

    Akos Koller

  3. Department of Physiology, University of Arizona, Tucson, AZ, USA

    Timothy W. Secomb

  4. Center for Cardiovascular Research, Charité Universitätsmedizin, Berlin, Germany

    Axel R. Pries

  5. German Heart Center, Berlin, Germany

    Axel R. Pries

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  1. Alex Marki
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  2. Eugeny Ermilov
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  3. Andreas Zakrzewicz
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  4. Akos Koller
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  5. Timothy W. Secomb
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  6. Axel R. Pries
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Correspondence to Axel R. Pries.

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Marki, A., Ermilov, E., Zakrzewicz, A. et al. Tracking of fluorescence nanoparticles with nanometre resolution in a biological system: assessing local viscosity and microrheology. Biomech Model Mechanobiol 13, 275–288 (2014). https://doi.org/10.1007/s10237-013-0499-7

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  • DOI: https://doi.org/10.1007/s10237-013-0499-7

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