Parameter

Description

Parameter Settings

Setting of alignment parameters.

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Registration Channel

Selects the registration channel. Use a channel with fiducials to which other channels will be registered to.
Note: A selection of more than one channel for the registration process is possible. For each selected channel an individual registration will be performed and, therefore, it is advisable to use one channel for the final registration. To test which channel will lead to the best results, all channels can be selected for later comparison of the results.

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Preregistration

Preregistration performs the process in a 2D maximum intensity projection of the z-stacks.
It is advisable to first perform a Pre-Registration to move similar structures, which might facilitate the Registration, already into close proximity.

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None

No Preregistration is performed.

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Hardware Rotation

The views are rotated solely based on the information from the rotation positioning of the sample holder which is saved in the metadata.

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Trans Hardware Rotation

The views are rotated solely based on the information from the rotation positioning of the sample holder which is saved in the metadata. Additionally, the x, y, and z axis are moved to match the views.

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Correlation Hardware Rotation

The views are rotated solely based on the information from the rotation positioning of the sample holder which is saved in the metadata. Additionally, the x, y, and z axis are moved to match the views. Cross-correlation is used to determine the translation in x, y, and z.

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Registration Method

Registration uses the complete 3D information of the z-stacks.

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Use Pre Register Only

No Registration process is performed and only the result of the Pre-Registration is used.

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Translation

The registration only corrects along the x, y, and z axis.

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Trans Rotation

The registration corrects along the x, y, and z axis and also the rotation along x and y.

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Dual side fusion

Merges the image done with the lightsheet from the right side with the image of the left side. See also Dual Side Fusion.

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Fusion Subset in X

Activated: Selects which area of the left and the right illumination image should be used for fusion. Only available for Mean or Maximum fusion. See also the description in Dual Side Fusion.

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Ignore Sample

Activated: Uses a function to find incorrectly labeled fiducials and eliminate them for registration.

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Expand to maximum value

Activated: The volume of the resulting dataset after registration and fusion is determined and additional black pixels (x,y) and z-slices are added to the views during registration. This prevents loss of data for later fusion.

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Scaling X=Y=Z

Activated: The dimensions are downsized to the smallest to have the same size. If the scaling in Deconvolution or PSF settings is changed, the latter overrides the scaling settings here.

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Individual Registration for every Time Point

Activated: Each time point generates a transformation matrix, and each time point is registered individually.
The time necessary for registration is multiplied by the number of time points and can increase substantially. However, if the motorized movement of the sample by the system has changed (e.g. due to thermic effects on the system: not fully warmed up at start, incubation, air conditioning of the room), the Individual Registration For Every Time Point option is necessary.

Deactivated: One transformation matrix is generated for all the following time points based on the first time point. As a result, processing for registration has only to be done once which reduces processing time.

Parameter

Description

Input settings

A subset of a present time series can be selected for the Registration and Fusion processing.

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Processing Subset Time

Truncates time points.

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Processing Subset Steps

Defines time-to-time point step size.

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Scaling correction (µm)

Sets the geometric scaling between X/Y and Z.

Fusion

Fuses registered views in one z-stack or one z-stack over time.

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None

No fusion is performed after Registration and Dual Side Fusion.

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Mean Fusion

The pixel in the fused image is determined by averaging the intensity level of the pixels for the involved views.

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Mean Fusion + Multiview DCV

Only available if the dataset has not undergone deconvolution before.
The pixel in the fused image is determined by averaging the intensity level of the pixels of the involved views. After fusion, a deconvolution is performed. The Deconvolution interface is displayed if selected.

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Blending

Controls the amount of Blending (smoothing) by fading the borders of all z-stacks in order to prevent visible edge effects.
Note: The value for Blending should be as low as possible with typical values from 20 to 80. This needs to be tested for every dataset.

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Fusion Subset in Z

Activated: Defines which portion of the z-stacks of the contributing views of the experiment are used for the fusion process.

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Current View

Selects the current view. For each view, the z-range can be set via slider, spin boxes, or mouse click in the image. Registration will not be affected by the z-range settings.
Note that for later deconvolution the calculated point spread function (PSF) of a Multiview experiment assumes that all views equally contribute to all areas of the image. This is not the case when using Subset Z and might result in artefacts of the deconvolution result.

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Save unfused data

Activated: Saves the registered image in addition to the fused image.