Quantification of labelled target molecules via super-resolution dSTORM localization microscopy

Dániel Varga; Hajnalka Majoros; Zsuzsanna Ujfaludi; Tibor Pankotai; Miklós Erdélyi

doi:10.1117/12.2545099

13 February 2020 Quantification of labelled target molecules via super-resolution dSTORM localization microscopy

Dániel Varga, Hajnalka Majoros, Zsuzsanna Ujfaludi, Tibor Pankotai, Miklós Erdélyi

Proceedings Volume 11246, Single Molecule Spectroscopy and Superresolution Imaging XIII; 1124612 (2020) https://doi.org/10.1117/12.2545099
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

The advent of super-resolution microscopy techniques has begun a revolution in the field of optical microscopy. Localization methods have become one of the most dynamically developed areas in this field. The one order of magnitude improvement in the lateral resolution has revealed a lot of new details mainly in the field of biology, which was hidden with conventional microscopes by the diffraction. However, the visualization of these measurements often go hand in hand with information loss and the quantification of such images become limited. Here we present a quantitative evaluation method which uses the raw localization coordinates and the associated precision for quantitative analysis. With our cluster analysis-based method we were able to determine the different properties of the selected clusters, such as their area (or in case of 3D images the volume), spatial distribution and the number of labelled target molecules in them, which is not follows directly from the reconstructed image due the stochastic nature of blinking and the often unknown labelling stoichiometry. The great advantage of our method is that we can gain the latter property from the sample directly. We also applied our method to investigate the repair mechanism of DNA double-strand breaks (DSBs).

Citation Download Citation

Dániel Varga, Hajnalka Majoros, Zsuzsanna Ujfaludi, Tibor Pankotai, and Miklós Erdélyi "Quantification of labelled target molecules via super-resolution dSTORM localization microscopy", Proc. SPIE 11246, Single Molecule Spectroscopy and Superresolution Imaging XIII, 1124612 (13 February 2020); https://doi.org/10.1117/12.2545099

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KEYWORDS

Molecules

Image segmentation

Switching

Microscopy

Point spread functions

3D acquisition

Computer simulations

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