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Proceedings Paper

Assessing resolution in live cell structured illumination microscopy
Author(s): Jakub Pospíšil; Karel Fliegel; Miloš Klíma
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Paper Abstract

Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology.

Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

Paper Details

Date Published: 1 December 2017
PDF: 7 pages
Proc. SPIE 10603, Photonics, Devices, and Systems VII, 106030D (1 December 2017); doi: 10.1117/12.2292793
Show Author Affiliations
Jakub Pospíšil, Czech Technical Univ. in Prague (Czech Republic)
Karel Fliegel, Czech Technical Univ. in Prague (Czech Republic)
Miloš Klíma, Czech Technical Univ. in Prague (Czech Republic)

Published in SPIE Proceedings Vol. 10603:
Photonics, Devices, and Systems VII
Karel Fliegel; Petr Páta, Editor(s)

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