STED microscopic parameters
- Load the image in Huygens Essential or Professional
- Right-click on the image and select "Parameter editor" or "Parameter wizard"
- Select "STED" as the microscope type to activate the specific STED microscopic parameters
- When using the parameter wizard, select "Expert&STED"
- If not all channels are STED:
- Select the deviating channel
- Check "deviating micr. type", and select the different microscope type
- Check and edit the "Excitation fill factor" and specific parameters for the STED channel with special care (see below).
Excitation Fill Factor - The ExcitationFillFactor is the ratio between the beam width and the diameter of the objective pupil. The Huygens default value is "2", meaning that the beam is twice as wide as the pupil. To improve energy efficiency, in the case of STED the fill factor was significantly lower. Previously, we recommend to use for STED an overfill factor of 1.2. With current STED systems a fill factor of 2.0 can be used. The default values are set accordingly.
STED depletion mode - This mode determines the type of STED depletion that is being used. You can set this to Pulsed, CW non-gated detection, or CW gated detection.
STED Saturation factor - The saturation factor (ς) is a factor describing the amount by which the fluorescence is suppressed by the STED beam. The higher this factor, the more suppression of fluorescence away from the optical axis, the more resolution. At values below 1 hardly any resolution is gained.
The saturation factor depends on factors like the laser power, gating timing in gSTED, and importantly the laser/dye combination. Typical values are in the range of 5 to 50.
For more details see this publication from the group of Prof. Stefan Hell.
STED wavelength - Enter here the wavelength of the STED depletion laser in nanometers.
STED Immunity fraction - This is the fraction of fluorescent molecules that is immune (photoresistant) to the depletion beam. The value that should be entered here is usually between 0% and 10%, and should be estimated with the use of the Huygens PSF distiller using STED bead images.
STED 3D- STED 3D microscopy uses orthogonal polarised beams to generate two coexisting depletion beams. The "donut" beam and an additional beam aimed at axial resolution improvement. Power may be shifted between the two beams to achieve the desired resolution. This parameter refers to the percantage of the used power dedicated to the Z "donut" beam.