™Huygens PSF Distiller
Distills a PSF from your bead images for monitoring microscope performance and improving deconvolution results
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. 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 easiliy compared to the theoretical (ideal) PSF or to experimental PSFs of microscopes from other labs.
No noise, no background
Reports Chromatic shift
Quality Assessment
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 DeconvolutionFast and easy with intuitive wizard
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 (Full Width Half Maximum) 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 is an indication of more image blurring.
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.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).
Sven Hildebrand, Anna Schueth, Klaus von Wangenheim et al., hFRUIT: An optimized agent for optical clearing of DiI-stained adult human brain tissue. PSF distiller and deskewing was used to restore Light Sheet data. Sci Rep 10, 9950 (2020).
For more, see Scientific PublicationsRelated products
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