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Huygens PSF Distiller

Measure your set-up specific PSF for quality assessment and optimal deconvolution results



With the PSF Distiller you can find the Point Spread Function (PSF) specific to your setup, which can be used to improve deconvolution results and to perform a quality assessment on your system. The Distiller wizard corrects for bead size and removes noise from your input bead images, making the Huygens measured PSF better suited for deconvolution than raw bead images.

A measured PSF is obtained by recording sub-resolution beads and loading the 3D images into the Distiller. The Distiller then corrects for the finite size of the beads, removes noise and, in the case of multiple beads, averages over all usable beads in the image. Since the resulting PSF contains aberrations specific to your system, which the ideal (model) PSF does not, it allows for the correction of such aberrations during deconvolution. The measured PSF also enables quality assessment of the microscope as it can be compared to the theoretical (ideal) PSF or to performance of microscopes from other labs.
PSF Distiller
Maximum Intensity Projections of nanobead STED (green) and confocal (red) image deconvolved with a theoretical PSF (left) or a measured PSF (right). The image deconvolved with the theoretical PSF does not show any separation between the two closely spaced beads (indicated by the arrow), while the measured PSF shows that it is possible to separate the two beads spaced at ~65 nm. Full figure.



No noise, no background

The Distiller puts out a noiseless PSF with no background signal, even from noisy images.


Reports Chromatic shift

Any chromatic shift is reported and can be corrected with the Chromatic Aberration Corrector.


Microscope Quality Assessment

Compare the measured with the theoretical PSF to check for setup-specific aberrations. Learn more





Image

About PSF distillation

A Point Spread Function (PSF) describes how an image is blurred by a microscope. The PSF can be distilled if both the true object and the resulting image are known. This is true for images of fluorescent beads with known dimensions. Once the PSF is distilled it can, in combination with the image, be used to restore images of which the true object is unknown through the process of deconvolution.

Learn more about image formation and Huygens True Deconvolution


Fast and easy with intuitive wizard

The PSF Distiller wizard performs the distillation in a few simple steps. The wizard can measure a PSF from one or multiple images, each containing one or more fluorescent beads. It is also possible to distill multi-channel PSFs from multi-color bead images, or to assemble a multi-channel PSF from separate single channel bead images’ PSFs.

The Distiller wizard’s unique features make the PSF distillation intuitive and easy.

  • Selects appropriate beads. The wizard uses a color coded image overlay to indicate which beads are okay and which have a deviant z position, a suspicious intensity, are located too far away from the optical axis or are too close to a neighbor or an edge. Only the okay beads are used for the distillation.

  • Accumulates and averages over beads. The wizard can accept multiple bead images at a time, extracting appropriate beads from each and accumulating them to average over. Note that for high SNR data, a single bead image may be sufficient for proper PSF distillation.

  • Automatically estimates FWHM. The PSF FWHM Estimator creates a line intensity profile through the center of the PSF in all three dimensions. For each, the width of the profile at half of the maximum intensity value is measured. The user can choose whether this is done linearly or through one of the available curve-fitting procedures. The FWHM quantifies microscope quality as a wider PSF gives more image blurring.
PSF Distiller Workflow
Bead data from Say-Tar Goh, CalTech, USA.




MeasuredVSTheoretical
At the top we see MIPs of a theoretical (left) and a measured (right) PSF for a widefield image. The measured PSF was distilled from an image of 170 nm beads, perfectly acquired (correct sampling rate, no clipping). Below we see line intensity profiles through the center of the theoretical (purple, dotted) and measured (yellow) PSF in each dimension. The three plots are not scaled the same.

Microscope quality assessment

Huygens PSF Distiller automatically allows for both qualitative and quantitative microscope quality assessment.

Every microscope is unique in that it has its own deviations from the ideal model: no two objectives are perfectly identical, alignments may vary, and so on. This has implications for the quality assessment and the reproducibility of resulting images. Therefore, in addition to information on sample preparation and acquisition conditions, a measure of the actual microscope quality is needed. The importance of including such a measure is illustrated by the formation and quick growth of the QUAREP-LiMi (Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy) initiative. The PSF provides a valid microscope quality measure as it shows how a point object is blurred by the microscope. The FWHM Estimator integrated in the Distiller wizard gives a quantitative measure of the quality; the output PSF allows for qualitative assessment, for example by comparing it with the theoretical PSF in all dimensions using the Twin Slicer.

We advise measuring the PSF after a change in the recording setup and certainly after each maintenance job in which the optics or scanning device was serviced. The measured PSF acts as a calibration of the microscope in the sense of relating a physical known object (bead) with what the microscope actually measures (bead image).



STED PSF

In the Huygens PSF distiller we integrated a robust STED thermal drift correction, which can automatically correct for the thermal drift that is often present in STED images. Moreover, at the end stage of the PSF distiller, the user can choose to estimate the STED parameters from the distilled PSF. An advanced iterative algorithm will estimate and fit the best theoretical STED imaging parameters with the distilled PSF, enabling a more robust theoretical PSF estimation that reflects the true imaging situation.

To measure STED PSFs, we recommend using origami nanocubes (for example these GATTAbeads). These sub-resolution objects (<40 nm for STED) can be packed with many dye molecules, making them better fit than the dim and bleaching-prone beads of similar size.
Image
3D stack of GATTA-Beads R measured at VUmc, Amsterdam, on a Leica TCS SP8 STED 3X. 3D PSF distillation and rendering by Scientific Volume Imaging.



Use in research

Emanuele Roscioli, Tsvetelina E. Germanova, Christopher A. Smith et al., Ensemble-Level Organization of Human Kinetochores and Evidence for Distinct Tension and Attachment Sensors.
PSF from the PSF Distiller was used for deconvolution of spinning disc images.
Cell Rep. 31 (4) (2020).

Pauline Dan, Eric Lin, Jingbo Huang et al., Three-Dimensional Distribution of Cardiac Na+-Ca2+ Exchanger and Ryanodine Receptor during Development.
PSF from the PSF Distiller was used for deconvolution of confocal images.
Biophys. J. 93, 2504-2518 (2007).

For more, see Scientific Publications

Any misalignment between 3D slices, for example due to sample stage instability or thermal fluctuations, should be corrected with Huygens' Object Stabilizer before PSF distillation. For STED imaging, after finding the experimental PSF with the Distiller's specialized STED settings, the image can be reliably deconvolved with Huygens STED Deconvolution.

Object Stabilizer STED Deconvolution

More information

Point Spread Function
Recording beads
Support page
PSF Distiller webinar


Figure5 Accumulutation To Distilled
Steps involved in PSF distilling. The Huygens PSF distiller can handle images that contain multiple beads (a). The distiller automatically selects the best beads by taking into consideration the size and separation of the objects in the images. These selected bead signals are accumulated. The accumulation process creates an average bead with higher SNR (b) which is beneficial to the distilling process. The distiller uses a non-linear algorithm to distill the PSF from the accumulated PSF (c). The distilled PSF is centered, normalized and free of noise, such that it can be used directly for deconvolution. All images are 3D MIP renderings in false color mode.

Figure8 ATTO647N Overview
Deconvolution results on the ATTO 647N nanobeads. All images are XY MIP projections. (a) raw dataset, imaged in confocal (red) and STED (green), (b) the same dataset deconvolved with the measured PSF, (c) zoomed-in region, deconvolved with theoretical PSF, (d) zoomed-in region, deconvolved with measured PSF, (e) intensity profile through the objects indicated by the arrow in (c) and (d). The image deconvolved with the theoretical PSF does not show any separation between the two closely spaced beads, while the measured PSF shows that it is possible to separate the two beads spaced at ~65 nm.

Figure7 FWHM Plots Overlayed
FWHM of theoretical STED PSF vs. measured (distilled) PSF for ATTO 647N beads. Intensity profile along X (a), along Y (b) and along Z (c) for the theoretical PSF (grey) and measured PSF (blue). The FWHM is also shown for each intensity profile. The theoretical PSF is calculated based on the STED parameters included in the Leica LIF meta-data. Notice that, especially at the base of the intensity profile, the measured (distilled) PSF is much narrower compared to the theoretical PSF, showing a narrower point-spread-function and a smaller FWHM in X and Y. Not final: STED laser was used at 50% of max. power.