Microscopic Parameters

The microscopic parameters are those describing the image and the acquisition optical conditions. The important ones to fully describe the Image Formation are:

Since Huygens 3.5 all microscopic parameters are set per channel, with the only exception of the geometrical ones that are common to all of them.

Global acquisition geometry parameters

• Sample Sizes in X, Y and Z.
• Coverslip Position (new in 3.5)

Per channel parameters

• Microscope Type
• Numerical Aperture
• Lens Refractive Index
• Medium Refractive Index
• Excitation Wavelength
• Emission Wavelength
• Back Projected Pinhole Radius
• Back Projected Pinhole Distance
• Excitation Photons

Since version 3.5, these parameters are also available (they will improve the calculation of a Theoretical Psf ):

• Excitation Fill Factor
• ObjectiveQuality

The algorithms also consider the Acquisition Depth .

For properly Doing Deconvolution the image must be correctly described by these parameters. They are specially necessary for calculating a theoretical Point Spread Function .

If values are displayed in a red background , they are highly suspicious (for example because a given Refractive Index is incompatible with that Numerical Aperture , or because the image is undersampled ). An orange background indicates a non-optimal situation.

OverSampling is also indicated with a cyan background, that becomes violet when it is very severe.

These parameters should not be tuned while Doing Deconvolution , 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. In the Huygens Software , the deconvolution tuning parameters are the Restoration Parameters .

Parameter templates

Microscopic parameters can be saved in Parameter Templates for later reuse.

In Essential

If you are following the Huygens Essential wizard please see the step → Check Microscopic Parameters .

Example

A typical example of microscopic parameters for a Two Channel image, with channels named Ch0 and Ch1:

x-y Sample Size (nm) 50
z Sample Size (nm) 150
Microscope Type Confocal Microscope
Numerical Aperture 1.0
Pinhole Radius (nm) 250 (Ch0) 275 (Ch1)
Lens Refractive Index 1.515 (Oil)
Medium Refractive Index 1.515
Excitation Wavelength (nm) 488 (Ch0) 543 (Ch1)
Emission Wavelength (nm) 519 (Ch0) 562 (Ch1)
Excitation Photons 1

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