STED parameters

If the selected microscope type is STED or STED 3D then a few STED parameters need to be specified to describe the STED acquisition. When working with other microscope types HRM will skip these parameters.

First, select the STED depletion mode: Pulsed, Continous wave gated detection, Continous wave non gated detection or Off/Confocal.

STEDModeScreenshot

Note

Notice that STED and confocal microscopy are often combined. That’s why even if the selected microscope type is STED it’s still possible to set specific channels as confocal at this point.

Set the STED saturation factor of each channel. Typical values are in the range of 10 to 50. The higher this factor, the more suppression of fluorescence away from the optical axis, the more resolution.

STEDSaturationScreenshot

Next, set the STED depletion wavelength of each channel. Notice that Channel 1 is grayed out in these figures because it was set as confocal.

STEDWavelengthScreenshot

The STED immunity fraction tells Huygens which percentage of the fluorescent molecules are not susceptible to the STED laser. Typical values are in the range 0% to 10%.

STEDImmunityScreenshot

Finally, depending on whether the microscope type is STED 3D or not, set the percentage of STED 3D per channel. This tells Huygens how much power was used in the Z depletion beam. The remaining power is used for the vortex path.

STED3DScreenshot