Spherical Aberration correction

Spherical aberration is mainly caused by a mismatch between the refractive indexes of the objective immersion medium and the sample embedding medium.

HRM will automatically detect this mismatch and ask whether the aberration should be corrected for as long as a theoretical PSF is used during deconvolution. This question is skipped otherwise.

Note

Spherical aberration can be corrected for by creating different theoretical PSFs (with slightly changing shapes) at different depths: depth-dependent PSF.

When using a depth-dependent PSF one also needs to specify whether the first plane in the dataset is closest or farthest to/from the coverslip.

The Correction mode allows for a more detailed choice as to how the depth-dependent PSF should vary with depth.

• Automatic correction: will generate a depth-dependent PSF where the PSF is automatically selected at each depth, as explained above.
• Advanced correction: allows to specify further details about the depth-dependent PSF.

The Advanced correction will further enable a number of advanced options.

• Depth-dependent PSF on few bricks: a different PSF will be generated for each Z brick in which the original data is split.
• Depth-dependent PSF slice by slice: a different PSF will be generated for each slice.
• PSF at user-defined depth: the user can define a specific depth for the generation of a PSF which will be used for all depths.

Note

Notice that splitting the original data into bricks while deconvolving not only helps to correct for spherical aberration but it also minimizes the amount of RAM memory needed for deconvolution.