A multi-photon microscope is a Fluorescence Microscope in which more than one Excitation Photons are used to excite the fluorophores.
The Multi-Photon Option in HuygensThe Multi-Photon option in the Huygens Software is required if you need to process multi-photon image data. It allows you to enter the number of photons involved in the fluorescence process as a parameter within the Microscopy Parameter Editor. The Multi-Photon option works in combination with the 'Widefield' and 'Confocal' microscope setting in the Parameter Editor, yet it does not require the presence of these optical options in the Huygens Software.
How to treat 2-photon data in HuygensBecause there are 'widefield' and 'confocal' multi photon microscopes, the Huygens 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 is handled differently by the Huygens software.
- different PSF generator, though it shares parts with the confocal generator.
- the reconstruct-PSF tool uses different a-priori knowledge about what the PSF should look 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 in your microscopic parameters (show via right-click on your image while in Huygens)
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.