Huygens Theoretical PSFA theoretical Point Spread Function (PSF) is a numerically computed PSF based on a model of the microscope and known Microscopic Parameters.
The Huygens Software uses advanced vector-based EM diffraction theory to generate the theoretical PSF including spherical aberration, which can subsequently be used for deconvolution. The Huygens PSF generator algorithm is fully multi-threaded, and is able to generate even very large PSF's very efficiently and can generate PSF's for a wide range of microscope models, including: widefield, confocal, 4pi, multi-photon, spinning-disk, STED and various light-sheet systems. In the near future we will also introduce PSF models for new microscope systems.
Refractive index mismatch distorts PSFWhen there is a refractive index mismatch between the lens medium and embedding medium, the PSF will become distorted. The amount of distortion will depend on the difference between the two refractive indices. In addition, the distortion will also depend on how deep you are imaging (with respect to the coverslip). The Huygens software can correct for spherical abberation when the microscopic parameters are set accordingly. The PSF generator takes into account: the coverslip position, imaging direction and RI of the lens and embedding medium (as read from the meta-data or set by the user). Huygens can use a varying PSF in the axial direction to accurately correct for the spherical aberration. With the unique varPSF option, Huygens can even apply multiple varying PSF's simultaneously during each iteration in the MLE algorithms for even more accurate spherical aberration correction.
Interactive example 1: Effect of embedding medium RI on PSF shape
change the embedding medium RI using the buttons
This interactive example shows a confocal PSF (XZ projection) generated by Huygens, with an NA of 1.4, and oil (1.51 RI) as lens medium at an acquisition depth of 0 (i.e. at the coverslip). Change the RI of the medium by clicking on the buttons to see the effect of the RI mismatch on the PSF shape.
Notice that between 1.51 (no RI mismatch), 1.45 and 1.40, not much is happening. However, once your embedding medium RI goes below 1.4 (the NA of the objective), the PSF is starting to change more significantly. This is because the NA of the objective will be effectively limited by the RI of the embedding medium in that case due to total internal reflection, which effectively will stretch the PSF along its axial axis.
Images affected by spherical aberration due to a refractive index mismatch are better restored with through the use of depth-dependent PSF's, because RI mismatch distorts the PSF. This depth-dependency can be achieved through theoretical PSF's calculating a different PSF for different acquisition depths. At this point Experimental PSFs can not be adapted for shperical aberration effects.
Interactive example 2: Effect of RI mismatch and imaging depth on PSF shape
change the acquisition depth using the buttons
This interactive example shows a confocal PSF (XZ projection) generated by Huygens, with an NA of 1.4, and oil (1.51 RI) as lens medium. The embedding medium is water (RI = 1.33). The refractive index mismatch between the lens immersion medium and the embedding medium will cause the PSF to distort: the PSF becomes more elongated and more assymetrical in the axial direction. The distortion becomes more severe when you are imaging deeper. Using the buttons above, you can change the depth of the imaging (with respect to the coverslip position), and inspect the effect of the imaging depth on the shape of the PSF.
Microscopy ParametersMany of the microscopic parameters can be read by Huygens directly from the raw file-formats. You can verify or adjust the parameters by right-clicking on the image thumbnail and then select 'Microscopic Parameters'. This will open the parameter editor (see image below), in which you can verify and/or adjust the relevant parameters, including refractive index and imaging depth. It is important to verify these parameters, so that the correct theoretical PSF will be used for deconvolution.
Light-Sheet PSFFor the various light-sheet models, Huygens generates PSF's that are also spacial-variant in the lateral direction (in the direction of the light-sheet), an effect caused by the non-uniform thickness of the light-sheet across the field of view.
Visualizating the theoretical PSFWith Huygens Professional you can calculate and generate a theoretical PSF image for any set of microscopic parameters using the "Theor.PSF" button in the taskbar of the Operation Window. Fore more detailed instructions, go to the Visualize Theoretical PSF tutorial page.
It is also possible to generate a PSF from the tcl command shell. For example to calculate and display the theoretical PSF of image "a" to thumbnail "psf" you run the follwoing command:
a genpsf -> psf