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Multiphoton microscope


A multi photon microscope is a Fluorescence Microscope in which more than one Excitation Photons are used to excite the fluorophores.

How to treat 2-photon data in Huygens


Mind that the multi-photon option in a License String doesn't activate a new Microscope Type: the number of photons involved in the fluorescence process is a Microscopic Parameter that you have to enter when describing the image. There are 'widefield' and 'confocal' multi photon microscopes, the multi photon option gives access to both. Two (or multi) photon data has more in common with confocal data than with wide field data, but still is handled differently.
For instance:

  • different PSF generator, though it shares parts with the confocal generator.
  • the reconstruct-PSF toold uses different a-priori knowledge about what the PSF should like
  • a number of basic values is computed differently, for instance the Nyquist rate, the expected two-point resolution, and so on.

For 2-photon microscopes, the Multi photon excitation parameter must be set to two.

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 set to Wide Field Microscope. 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 500 nm Back Projected radius, it must be taken into account. So in this case the microscope type type should be set to confocal, and the correct Back Projected Pinhole Radius specified.


SHG signal of the rat soleous muscle's myosin imaged with a Leica two-photon microscope (NA: 1, 20x). Stack has been deconvolved with Huygens using a measured PSF, and visualized with the Huygens SFP Renderer. High quality imaging together with Huygens allows you to see through the muscle's A bands from one side to the other. The stack dimensions is 105x105x87 μm.
SHG signal of the rat soleous muscle's myosin imaged with a Leica two-photon microscope (NA: 1, 20x). Stack has been deconvolved with Huygens using a measured PSF, and visualized with the Huygens SFP Renderer. High quality imaging together with Huygens allows you to see through the muscle's A bands from one side to the other. The stack dimensions is 105x105x87 μm.

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