Huygens Localizer
curves is to localize the microspheres and fit a 2D Gaussian shape at each Z position, followed by fitting defocusing curves to the resulting heights and widths [10]. Huygens Localizer improves on this approach by offering the option to fit a spline function for cases where the measured data deviate too much from a theoretical defocusing curve. Besides fitting the microspheres directly, Huygens Local-
izer also offers the option to estimate the PSF using the Huygens PSF Distiller module. With the distiller module, an accurate, nearly noise-free PSF can be estimated from multiple micro- sphere images. Te distilled PSF can then be used to calculate high-quality Z calibration curves that can be used instead of
Figure 5: Analysis of 2D SMLM data. (A) Sum projection of all images in the SMLM time-series, corresponding to a wide-field image. Scalebar: 5 μm. (B) High-resolution rendering of the SMLM localization results of Huygens Local- izer. (C) High-resolution rendering of an area in (B) indicated by the white box. Scalebar: 0.5 μm. (D) A plot of the intensities along the line indicated in (C). The full width at half maximum is indicated in red and has a value of 55 nm.
Analysis of SMLM data. Figure 2 shows the steps followed
by Huygens Localizer to turn a series of SMLM images into a table of localizations and a high-resolution image. Te stages of the analysis pipeline include:
1. Background detection: Huygens Localizer implements a time-based background estimator that separates static structures from blinking fluorophores.
2. Detection of the single particles: Te particles are detected in each frame by looking for local intensity maxima exceed- ing a threshold.
3. Localization of the detected particles: Each localization is fitted using maximum likelihood estimation, least-squares fitting, or center-of-mass determination. In 3D SMLM, the Z positions of the particles are also accurately calibrated.
4. Filtering of unwanted particles: Huygens Localizer imple- ments several filters to interactively remove particles from the table of localizations.
5. Driſt correction: Huygens Localizer offers an automatic driſt corrector that does not require fiducial markers, avoid- ing the burden of introducing physical markers during sample preparation.
6. Visualization: Particles are rendered on the fly into a high- resolution image as single points or by placing small 2D/3D Gaussian spots at each particle location.
In each of these steps Huygens Localizer leverages parallel computing on multi-core CPUs and GPUs in order to perform these calculations as quickly as possible. Importantly, this allows interactive adjustment of crucial parameters, such as the thresholds that affect particle detection. Calibration of the Z position. Calibration is generally
done by imaging microspheres (∼100 nm) at small axial inter- vals (∼10 nm). Te conventional approach to create calibration
2020 March •
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Figure 6: Drift correction for SMLM data. (A) Rendering of SMLM localizations without correction for drift. Scale bar: 2 μm. (B) Rendering of SMLM localiza- tions after corrections for drift using the Huygens Localizer drift corrector. The white arrows show a vertical double structure that is preserved by the drift corrector, in contrast to the horizontal double structures that are caused by drift. (C) Screenshot of the drift corrector, showing a plot of the shifts in the horizontal (red) and vertical (green) directions as a function of frame number in the time series.
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