Authors:
Stefan Sokoll
1
;
Klaus Tönnies
2
and
Martin Heine
1
Affiliations:
1
Leibniz Institute for Neurobiology, Germany
;
2
Otto-von-Guericke University, Germany
Keyword(s):
3D particle localization, Nanometer sub-resolution, Live cell imaging, Confocal fluorescence microscopy.
Related
Ontology
Subjects/Areas/Topics:
Algorithms and Software Tools
;
Bioinformatics
;
Biomedical Engineering
;
Computational Molecular Systems
;
Image Analysis
;
Model Design and Evaluation
;
Pattern Recognition, Clustering and Classification
Abstract:
Studying molecular dynamics is crucial for understanding biological processes in living cells. In principle, this is achieved by attaching fluorescent particles to molecules of interest and their detection using fluorescence microscopy. These analysis require fast optical techniques with at least 20Hz frame rate and a resolution below the diffraction limit in all three spatial dimensions. Current approaches basically rely on determining the correlation between features of the particle’s 2D point spread function (PSF) and the focal distance to the center of the particle. However, they are still unsuitable for the application to live cell imaging where the
refractive index mismatch is present. This mismatch leads to non-stationary optical properties of the particles on which the algorithms rely, necessitating a calibration procedure prior to every experiment. However, this is almost unfeasible to particles attached to living cells.
We established a spinning disk confocal setup and empl
oy Quantum dots (QD) as fluorescence particles. Corresponding models of the axial PSF features that define the distance to the center of the particle are developed and analyzed in the presence of the refractive index mismatch. We present this analysis as the base for the future development of a 3D localization technique applicable to living cells.
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