PSF Distiller

By default, Huygens uses a theoretical PSF to deconvolve your image data, so no extra work is needed. When a deconvolution run is started the theoretical PSF will be automatically calculated from the microscopy parameters and used "on the fly".

Huygens also offers the option to load and use an experimental PSF, and is optionally equipped with the PSF Distiller. This wizard driven tool guides the user through the process of distilling a Point Spread Function (PSF) from 3D bead images. It helps in selecting bead images, and in creating and saving the PSF for further deconvolution runs. The wizard is able to measure a PSF from one or multiple images, each containing one or more fluorescent beads. It is also possible to distill multi-channel PSF's from multi-color bead images, or to assemble a multi-channel PSF from separate single channel bead images PSF's.

Measured PSF's improve deconvolution results and may also serve as a quality test for the different supported microscopes. We advise to measure 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).

¹ TetraSpeck™ microspheres by Thermo Fisher Scientific.
² GATTA-Beads by GATTAquant GmbH.

STED PSF

For accurate STED PSF measurement, you need beads smaller than 40nm, and ideally around 20nm in size. Unfortunately most beads of this size are very dim and may photobleach very quickly, making it practically impossible to measure a z-stack for 3D PSF measurement. As an alternative, for STED we advised to use GATTAbeads (GATTAquant GmbH., Germany), which are DNA origami objects folded as nanocubes and are packed with fluorophores. This efficiently creates spherical ‘beads’ with a size of approx. 23nm * 23nm * 40nm. These type of beads have shown to be relatively bright and photostable. In addition, these GATTAbeads can be customized with any type of organic dye and embedding medium, so that it the measured PSF reflects the actual PSF in your sample as best as possible. In the HUYGENS PSF distiller we integrated a robust STED thermal drift correction, which can automatically correct for thermal drift which 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.