Multi-Dimensional Open Dialog

The Multi-Dimensional Open Dialog controls loading individual x,y,t image series from multi channel, position and x,y,z,t recordings, from the following formats:
Metafluor *.inf
Metamorph *.nd
Nikon Elements *.nd2
Zeiss LSM Multi Time Series: *_Sum.lsm
TIF files arbitrarily organized in folders
BD pathway Experiment.exp as TIF folder
Any other formats using the Bio-Formats reader

In the Open dialog multiple files can be selected, these files will be merged in time or as channels or as positions depending the settings in the Multi-Dimensional Open Dialog. When merging in time, the time passed between recordings is taken into account, whenever possible.

To load an image series as is, in the Multi-Dimensional Open Dialog:

1. Select stage position
2. Select which channels or wavelength to load
3. Select 'None' in Processing
4. Press Open or press Open Next to load the next stage position

Z-projection:
x,y,z,t image sequences (stacks) are loaded as Z-projected x,y,t image sequences (stacks), see Settings tab.
To load one time frame of a x,y,z,t data set non-projected select the Single Time Point in the Processing box in the Open tab.
Z-projection is performed during loading, and only the projection images are stored to reduce memory usage. 

Dehazing (or blind deconvolution):
To load x,y,z,t data set dehazed (blind deconvolved) use the Spatial filtering option. Each plane of x,y,z,t data sets is separately filtered with the specified spatial filter. Filters have to be specified separately for each channel. Z-projection is performed on the filtered z-stack. The filtering is performed during loading. Filter files can be generated and saved using the Tools/'Setup DFT filter and Filter Optimizations' dialog.

Optical flow (OF) load requires and an x,y,t,t data set, e.g. a time lapse of short time lapses. Because this is not supported natively by most acquisition environments, the following tricks are available (see using optical flow in details in our protocols):

1. In most acquisition environments a time lapse of short time lapses can be recorded as  x,y,z,t stacks, just the z-step is set to zero and a certain delay is applied in between acquisitions. For optical flow load select only the channel that contains x,y,t,t data, and see the Optical flow tab for further settings.
2. A time lapse can be separated to blocks, and the time lapse of these blocks can be handled as x,y,t,t data. This is typical for Zeiss Multi Time Series data. To enable this feature see Block mode of the time lapse in the Settings Tab.
3. A time lapse of short time lapses can be generated by recording multiple channels with the same settings with time delay. This is typical for Nikon Elements data, see below, ND2 Tweak tab, Load multiple channels as Z or OF.

Merging data files:
Use either multiple file selection in the Open dialog, or drag multiple files from the Windows explorer onto the Image Analyst MKII. In addition you can add, remove and reorder files use the File Order tab of the Multi-Dimensional Open Dialog.

• To merge experiments in time, data sets must have the same x,y dimensions, and have the same number of stage positions and channels, though they don't have to have the same z-dimension. The time passed between recordings is taken into account, whenever possible.
• To merge as channels, select this option in the File merge box in the Open tab. The number of frames in each file that are merged as channels needs to be the same. Channels corresponding to each file will be listed in the 'Select channel' list.
• To merge as positions, select this option in the File merge box in the Open tab. All positions from all files will appear in the positions list, and available for pipeline automation.

Open (main) tab:

TIF Folder tab: (only for opening TIF folders)

• The TIF Folder tab allows to explore and read files in a single folder or in a folder structure as a multi-dimensional data set.
• Select rule or pattern
• Press Scan to explore files
• Read more about setting up rules or patterns for organizing TIF files into a multi-dimensional data set.

File (or Dataset) Order tab:  

• To add, remove and reorder files use the File Order tab.
• When leaving the File Order tab, the files are re-scanned so switching between the Open and File Order tabs can be used to update files when looking e.g. nd2 currently recorded with the image acquisition software.

Settings tab:

• Project Z: set here how to project x,y,z,t datasets during loading
• Load specified frames only of each stack: the Z-projection or Optical Flow Load will take only the specified frames in account
• Use z-drift stabilizer: Enables the Z-Stabilizer tab for sub-stack projection centered around a frame that is chosen based on similarity to the previous or first time point of the time lapse.
• Load specified frames only of the time lapse: only the specified frames of the complete time lapse (considering all frames of the merged image sequences) will be loaded. Use this setting to override improperly stored frame number information when analyzing nd2 files during acquisition (of the same file) with Nikon Elements. To this end check the 'Forced loading of ND2 frame numbers'.
• Block Mode of the Time Lapse: use this settings  if the time lapse was recorded by the block feature of the Zeiss LSM software Multi Time Series module. Generally, this considers (for any file format) that the time lapse is a repeating sequence of blocks of certain number of frames. Each block contains one or more sequence of frames, which will be handled as separate channels. Enter the number of frames for each sequence within the block, separated by commas. In the Open tab each channel will be split according to the number of sequences. When using *.lsm files, these sequences can be used for Optical flow calculation by enabling Treat as Z or OF checkbox. See details in the corresponding protocol.
• Short Image Window names: (Metamorph specific) Only the base name and stage number is shown in the Image Window caption for ND files.
• Ignore STK file time stamps:  (Metamorph specific) Uses time information encoded in standard tif-tags instead of the Metamorph-specific time information. This is rounded to seconds, and less accurate than the Metamorph-specific time information. Use it only if encountering problems, e.g. when an stk does not contain time lapse information.

Plate tab

• When multi-position data sets are opened the dialog will attempt to identify a well name or position number within the position name, if such is provided by the data sets or the metadata within.
• Well names and position numbers are essential for ordering results in microplate format in the Excel Data Window.
• Read more about how to adjust detection of well names.

ND2 Tweak tab (shown only for Nikon Elements *.nd files):

• Load Speed: The default option is to Keep ND2 files open as long as the Multi-Dimensional Open Dialog is open to avoid repeated reading of metadata. Cache stage position caches image data in between reading of different channels of a given stage position. When reading is finished, the cache is emptied. Disable this function if running out of memory when using nd2 files. Useful if having enough RAM and reading nd2 files of several gigabytes. Keep cache until OF load: use this feature if performing OF load after non-processed load of the same stage position to speed up OF calculation.
• Safe mode can be used to read broken nd2 files.
• Tweaks for Optical Flow: OF calculation needs very accurately recorded time points. Nikon Elements records one time point per lambda cycle, therefore in z-stacks only the first image is timed properly, and time spent between recording of separate channels is not resolved. To assign time point to each image Image Analyst MKII reads the event information from nd2 files. An event can be any macro command, or comment, e.g.: /*time stamp*/. The same macro command (or the same comment within a command) has to be assigned in the Nikon Elements ND acquisition dialog Advanced section to all channels or to all z-planes. In the Image Analyst MKII the The available event markers are listed in the Tweaks for Optical Flow panel. Checking the Use events checkbox and selection of an event results that the timing of each frame will be obtained from the timing of the corresponding event.
• Load multiple channels as Z or OF: OF can be calculated from lambda cycles recorded by Elements, if the timing is obtained from an event associated with each channel of the  lambda cycle,  and no Frames of each Stack or Block mode are selected in the Settings tab.

Bio-Formats Tweak tab

Select different time stamp order if time stamps are loaded incorrectly.

Spatial Filter tab:

To load spatially filtered images (Z-stacks are filtered before projection):

1. Check 'Use Spatial Filtering'
2. Set the total Number of channels
3. Define 2D DFT filter for each channel by using the Select Channel and by setting the parameters of the filter. This filter works identically to the 2D DFT filter, see the parametering there, See also how to design filter functions and the spatial filter topics.
4. Press Open.

OpticalFlow Load tab (go to the protocols page for specific instructions on using this feature):

To load an x,y,t,t data set as time lapse of Optical Flow images:

1. In the Open tab select the channel containing the x,y,t,t data set
2. Alternatively select the (identically recorded) channels containing the x,y,t,t data set
3. In the OpticalFlow Load tab check the 'Optical Flow (OF) Load'
4. Spatial filtering is generally not required for Optical Flow calculation, but spatial filtering can precede optical flow calculation by checking 'Use spatial filtering'.
5. Set parameters as described at the Optical Flow function.
6. If the 'Output as Absolute value of Projected Vectors' is set to True or a Background ROI is needed to be defined, load the image set first with Processing set to None in the Open tab, then define ROIs, and in the OpticalFlow Load tab parameter list refer to the ROI numbers in that (last opened) Image Window. Switch Processing back to Optical Flow.
7. Press Open.