The recorded views of an experiment are registered manually. The views of one channel at one time point of the experiment are the basis for the procedure. The z-stacks of the individual views are rotated based on the rotation information of the files metadata and transformed into channels, resulting in one maximum intensity projection image. Within this newly generated image, the views, now behaving as channels, can be moved in x, y, z and rotated to overlay each other. When all views are laid on top of each other, the Apply button uses the adjusted parameters to register, and when required to fuse, the views for all time points of the experiment. An .xml file is created which can be used for the Use registration from file option. This .xml file can be found along with the processed data in the result folder.

Parameter

Description

Parameter Settings

Setting of alignment parameters.

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Registration Channel

Selects the registration channel that should be used to manually overlay all views of the image.
Note: Only one channel can be used for this procedure, all other channels will not be displayed.

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Current Time Point

Indicates the selected time point of a time series as set with the time slider in the Dimensions tab.

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Front View

Converts views into channels and generates a maximum intensity projection along the z-axis of channels displaying the x- (horizontal) and y- (vertical) axis of channels representing.
Note: It is advisable to assign different colors to the channel to ease up alignment process. Channels can be turned on/off in the Dimensions tab.

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Side View

Converts views into channels and generates a maximum intensity projection along the x-axis displaying the z- (horizontal) and y- (vertical) axis of channels representing.
Note: It is advisable to assign different colors to the channel to ease up alignment process. Channels can be turned on/off in the Dimensions tab.

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View #

Selects the view to be aligned to View 1.
View 1 is the master view and the reference channel and cannot be selected or moved. View # can be moved by translation and rotation against View 1. It does not matter if alignment is started with Front View or Side View. Three iterations between Front View and Side View alignment are recommended.

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Alignment X

Shifts the selected view against View 1 in X. Not available for Side View.

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Alignment Y

Shifts the selected view against View 1 in Y.

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Alignment Z

Shifts the selected view against View 1 in Z. Not available for Front View.

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Rotation

Rotates the selected view around the position of the red cross overlay within the image of the 2D tab.

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Reset

Moves all views back to the position where the maximum intensity projection was generated.

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Preview

Produces a new image of the channel and the time point which is registered with the provided settings. The image will contain fused views when Mean Fusion is selected for Fusion.

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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 is used for fusion. Only available for Mean or Maximum fusion. See also the description in Dual Side Fusion.

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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, PSF settings is changed, the latter will override the scaling settings here.

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.