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Proceedings Paper

Localization of single biological molecules out of the focal plane
Author(s): L. Gardini; M. Capitanio; F. S. Pavone
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Paper Abstract

Since the behaviour of proteins and biological molecules is tightly related to the cell’s environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.

Paper Details

Date Published: 4 March 2014
PDF: 8 pages
Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 895019 (4 March 2014); doi: 10.1117/12.2039369
Show Author Affiliations
L. Gardini, European Lab. for Non-linear Spectroscopy, Univ. degli Studi di Firenze (Italy)
M. Capitanio, European Lab. for Non-linear Spectroscopy, Univ. degli Studi di Firenze (Italy)
F. S. Pavone, European Lab. for Non-linear Spectroscopy, Univ. degli Studi di Firenze (Italy)
Istituto Nazionale di Ottica (Italy)

Published in SPIE Proceedings Vol. 8950:
Single Molecule Spectroscopy and Superresolution Imaging VII
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

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