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

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 7μm) 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 6.5μm 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 0.8μm 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.