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Huygens Microscopic Parameters Editor

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FAQ


General

The Microscopic Parameter Editor in Huygens Essential, Professional and Localizer is a tool that can be used to view, edit and verify microscopy parameters of images. For more information about microscopy parameters visit the Huygens Imaging Academy
You can edit the microscopic parameters of an image in Huygens by selecting the image and open the Microscopic Parameter Editor via the top menu in Huygens or right-click on the image and selecting the Microscopic Parameter editor in the quick menu.

Resolving warnings & Verifying parameters

It is not strictly needed, but highly recommended to verify the microscopy parameters of an image before doing analysis, visualization or restoration like deconvolution. In some cases, the acquisition parameters are not properly saved by the microscope software. In addition, values such as fluorophore excitation and emission wavelengths, refractive index values of the sample medium, and the position of the Z stack with respect to the coverslip are typically not registered by the hardware and thus not saved with the image metadata. Therefore, we recommend users to verify all the microscope parameters in Huygens. Correct microscopy parameters ensure better and reliable result.
The different background colors of the input fields warn that there is potentially something wrong with the parameter value. In the reports section of the Parameter Editor window, you can find an explanation about the warning and suggestions on how to resolve the issue. The colored dots infront of each parameter, show the status of the parameter. When hovering with your mouse on a dot you will get a short explanation of the status. For a precise explanation of the meaning of each color see the Huygens Imaging Academy.
It is important to note that the parameters values should not be changed to prevent warnings, they should simply describe the real acquisition conditions. If you get warning messages about undersampling for example, nothing will be gained by changing these parameters to avoid them. By doing so, you can produce even worse restoration results.

Parameter values

A complete overview of all microscopy parameters is given in on our Huygens Imaging Academy webpage. You are able to find more information on each parameter there, as well as information on how to determine the correct value for each parameter.
The backprojected pinhole calculator on our website can be used to determine the parameter value for different microscopes.
With the Nyquist Calculator the Ideal Sampling from your optical conditions can be determined. The ideal Sampling Density (or inversely, Voxel Size) for image acquisition depends on the optics of the microscope. It is recommended to sample the image at a rate close to the ideal Nyquist Rate. Images obtained with sampling distances (voxel dimensions) larger than those established by this rate will suffer from undersampling. See the examples on anti aliasing and aliasing artifacts, and some consequences in Quality Vs Sampling.
Very large or no pinhole
If there is a non-descanned detector (NDD), or a very large (say > 10 Airy disks) pinhole, the contribution of the pinhole to the image formation can be ignored. In this case the microscope type can be widefield in combination with 2p photon. This will result in more realistic values for the Nyquist rate than a confocal setting.

Fairly large pinhole
If there is a pinhole in the order of a couple of Airy disks, typically 500nm backprojected radius, it must be taken into account. So in this case the microscope type should be set to confocal.


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