This method allows you to use a multi-channel image, typically an image representing the different colors you aim to image, to create a transformation file for channel alignment. It is best to use multicolor beads of the size which correspond to the used objective, for example, use 100 or 120nm beads when working with an objective with high numerical aperture like a 63x oil objective, to visualize in detail potential chromatic shifts. This file can be used to align channels in your image, see Apply Channel Alignment Transformation. Additionally, this function creates an output image where the channels are aligned with the estimated transformation parameters. This method is especially aimed at imaging multicolor beads not at extended biological structures. As an input you can use a multi-channel image or multi-channel z-stack, other dimension like tiles are not supported. Note that SIM and Airyscan images have to be processed first.

Parameter

Description

Output File

Defines the name and path of the output xml file.

Alignment Quality

Selects the alignment quality. The higher the selected quality, the slower but more robust and accurate the alignment will be. This is mostly relevant for large z-stacks. For 2D images the function should be fast enough even with the highest quality setting.

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Low

Uses the fastest setting, which can be especially useful for large images containing dense biological structures.

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Medium

This is the default setting for bead measurements with normal density.

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High

This setting is for bead measurements if higher accuracy is required.

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Highest

This is the most accurate setting. It is recommended if High does not deliver a good result and if there is a large z-mismatch of channels.

Reference Channel

Selects the reference channel to which other channels will be aligned. If alignment proves difficult, select the channel with the best signal to noise ratio or the one that has the most overlap with the other channels.

Adjust per Channel

Activated: Opens a list with the channels to allow an individual adjustment of each channel. For every channel you have the following options:

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Process Channel

Aligns this channel according to the parameters.

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Skip Channel

Skips this channel when processing. This channel will not be in the output image.

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Copy Channel

This channel is copied into the output image without alignment.