With this method you can create shading reference images for multi-channel tile images. It collects the intensity information from many tiles and creates an averaged image representing the shading influences of the system on the image. Note: Z-stack and time series images cannot be processed with this function! Best results are achieved for images containing more than 200 tiles per channel. The software creates the shading reference image and stores it directly to the Calibration Manager (Shading Reference).

Parameters

Description

All Scenes

Only available for multi-scene images.
Selects all scenes for processing.

Selected Scene

Only available for multi-scene images and if All Scenes is deactivated.
Selects the scene used for processing.

Merge Channels

Creates a single multi-channel-reference-image (with the same channel order) in one step.

Adjust per Channel

Only available for images with multiple channels.

Activated: You can adjust the settings for every channel separately. To use a channel, select Process Channel or select Skip Channel if you do not want a channel to be processed.
The settings Save directly as Shading Reference and Channel-specific are applied separately for each channel. If you want to use the same settings for all channels, deactivate the checkbox.

Channel-specific

Activated: The software performs channel-specific shading correction. In this case the fluorescence filter used during image acquisition is saved with the shading file. The following components will be included in the shading reference and checked before applying shading correction:
Contrasting method and condenser, fluorescence filter, magnification: Objective and Optovar; camera bit depth and RGB/BW mode, camera type and port position.

Deactivated: The system creates an All Channel calibration and perform an objective specific shading correction. The following components will be included in the shading reference and checked before applying shading correction:
Magnification (Objective and Optovar); Camera bit depth and RGB/BW mode, and camera type and port position.

Auto Adjust Intensity

Activated: Automatically calculates the multiplication factor based on the gray values of the image and the needed gray values for using it in the shading reference calibration manager.
If activated, the setting for Multiply Factor has no influence anymore on the image generation.

Multiply Factor

Only available if is Auto Adjust Intensity deactivated. Sets a multiplication factor, i.e. the software will multiply the pixel intensity for each pixel of the shading reference image by this value.
If you use an own sample, it is mostly the case that the images are very dim and the intensity does not reach the value needed to be used within the shading reference calibration manager, thus it will be rejected.

Apply Gaussian Filter

Activated: A Gaussian filter is applied after the averaging of the field of views from a tiled image is done. This enables to smooth the shading reference image.
Use this filter and the Sigma factor very carefully as it could remove also features which are real shading structures. This feature could be recommended if the number of tiles in the scanned image is low and cannot be increased for certain reasons.

Sigma

Only available if Apply Gaussian Filter is activated.
Defines the strength of the smoothing.