In this context, the term image sequence is used to refer to a collection of images from a time-lapse assay (movie), a three-dimensional (3-D) Z-stack assay, or both. This section will teach you how to load these collections in order to properly represent your data for processing.

## Sequences of individual files¶

For some microscopes, the simplest method of capturing image sequences is to simply acquire them as a series of individual image files, where each image file represents a single timepoint and/or Z-slice. Typically, the image filename reflects the timepoint or Z-slice, such that the alphabetical image listing corresponds to the proper sequence, e.g., img000.png, img001.png, img002.png, etc.

It is also not uncommon to store the movie such that one movie’s worth of files is stored in a single folder.

Example: You have a time-lapse movie of individual files set up as follows:

• Three folders, one for each image channel, named DNA, actin and phase.

• In each folder, the files are named as follows:

• DNA: calibrate2-P01.001.TIF, calibrate2-P01.002.TIF,…, calibrate2-P01.287.TIF
• actin: calibrated-P01.001.TIF, calibrated-P01.002.TIF,…, calibrated-P01.287.TIF
• phase: phase-P01.001.TIF, phase-P01.002.TIF,…, phase-P01.287.TIF

where the file names are in the format <Stain>-<Well>.<Timepoint>.TIF.

• There are 287 timepoints per movie, and a movie of the 3 channels above is acquired from each well in a multi-well plate.

In this case, the procedure to set up the input modules to handle these files is as follows:

• In the Images module, drag-and-drop your folders of images into the File list panel. If necessary, set your rules accordingly in order to filter out any files that are not part of a movie sequence.

In the above example, you would drag-and-drop the DNA, actin and phase folders into the File list panel.

• In the Metadata module, check the box to enable metadata extraction. The key step here is to obtain the metadata tags necessary to do two things:

• Distinguish the movies from each other. This information is typically encapsulated in the filename and/or the folder name.
• For each movie, distinguish the timepoints from each other and ensure their proper ordering. This information is usually contained in the filename.

To accomplish this, do the following:

• Select “Extract from file/folder names” or “Import from file” as the metadata extraction method. You will use these to extract the movie and timepoint tags from the images.
• Use “Extract from file/folder names” to create a regular expression to extract the metadata from the filename and/or path name.
• Or, use “Import from file” if you have a comma-delimited file (CSV) of the necessary metadata columns (including the movie and timepoint tags) for each image. Note that microscopes rarely produce such a file, but it might be worthwhile to write scripts to create them if you do this frequently.

If there are multiple channels for each movie, this step may need to be performed for each channel.

In this example, you could do the following:

• Select “Extract from file/folder names” as the method, “From file name” as the source, and .*-(?P<Well>[A-P][0-9]{{2}})\.(?P<Timepoint>[0-9]{{3}}) as the regular expression. This step will extract the well ID and timepoint from each filename.
• In the new group of extraction settings, select “Extract from file/folder names” as the method, “From folder name” as the source, and .*[\\/](?P<Stain>.*)[\\/].*\$ as the regular expression. This step will extract the stain name from each folder name.
• Click the “Update” button below the divider and check the output in the table to confirm that the proper metadata values are being collected from each image.
• In the NamesAndTypes module, assign the channel(s) to a name of your choice. If there are multiple channels, you will need to do this for each channel.

For this example, you could do the following:

• Select “Assign images matching rules”.
• Make a new rule [Metadata][Does][Have Stain matching][actin] and name it OrigFluor.
• Click the “Add” button to define another image with a rule.
• Make a new rule [Metadata][Does][Have Stain matching][DNA] and name it OrigFluo2.
• Click the “Add” button to define another image with a rule.
• Make a new rule [Metadata][Does][Have Stain matching][phase] and name it OrigPhase.
• In the “Image set matching method” setting, select “Metadata”.
• Select “Well” for the OrigFluor, OrigFluo2, and OrigPhase channels.
• Click the button to the right to add another row, and select “Timepoint” for each channel.
• Click the “Update” button below the divider to view the resulting table and confirm that the proper files are listed and matched across the channels. The corresponding well and frame for each channel should now be matched to each other.
• In the Groups module, enable image grouping for these images in order to select the metadata that defines a distinct movie of data.

For the example above, do the following:

• Select “Well” as the metadata category.
• The tables below this setting will update themselves, and you should be able to visually confirm that each well is defined as a group, each with 287 frames’ worth of images.

Without this step, CellProfiler would not know where one movie ends and the next one begins, and would process the images in all movies together as if they were a single movie. This would result in, for example, the TrackObjects module attempting to track cells from the end of one movie to the start of the next movie.

If your images represent a 3D image, you can follow the above example to process your data. It is important to note, however, that CellProfiler will analyze each Z-slice individually and sequentially. Whole volume (3D image) processing is supported for single-channel .TIF stacks. Splitting image channels and converting image sets into .TIF stacks can be done using another software application, like FIJI.

## Basic image sequences consisting of a single file¶

Another common means of storing time-lapse or Z-stack data is as a single file containing frames. Examples of this approach include image formats such as:

• Multi-frame TIF
• Metamorph stack: STK
• Evotec/PerkinElmer Opera Flex
• Zeiss ZVI, LSM
• Standard movie formats: AVI, Quicktime MOV, etc

CellProfiler uses the Bio-Formats library for reading various image formats. For more details on supported files, see this webpage. In general, we recommend saving stacks and movies in .TIF format.

Example: You have several image stacks representing 3D structures in the following format:

• The stacks are saved in .TIF format.
• Each stack is a single-channel grayscale image.
• Your files have names like IMG01_CH01.TIF, IMG01_CH02.TIF, … IMG01_CH04.TIF and IMG02_CH01.TIF, IMG02_CH02.TIF, etc, where IMG01_CH01.TIF designates channel 1 from image 1, IMG01_CH02.TIF designates channel 2 from image 1, and IMG02_CH01.TIF designates channel 1 from image 2.

You would like to process each stack as a single image, not as a series of 2D images. In this case, the procedure to set up the input modules to handle these files is as follows:

• In the Images module, drag-and-drop your folders of images into the File list panel. If necessary, set your rules accordingly in order to filter out any files that are not images to be processed. In the above example, you would drag-and-drop the .TIF files into the File list panel.
• In the NamesAndTypes module, select “Yes” for “Data is 3D”. You should also provide the relative X, Y, and Z pixel sizes of your images. X and Y will be determined by the camera and objective you used to capture your images. Your Z size represents the spacing of your Z-series. In most cases, the X and Y pixel size will be the same. You can divide the Z size by X or Y to get a relative value, with X = Y = 1. CellProfiler will use this information to correctly compute filter sizes and shape features, for example. Additionally assign each channel to a name of your choice. You will need to do this for each channel. For this example, you could do the following:
• Select “Assign images matching rules”.
• Make a new rule [File][Does][Contain][CH01]
• Provide a descriptive name for the channel, e.g., DAPI.
• Click the “Add another image” button to define a second image with a set of rules.
• Make a new rule [File][Does][Contain][CH02]
• Provide a descriptive name for the channel GFP.
• Click the “Update” button below the divider to confirm that the proper images are listed and matched across the channels. All file names ending in CH01.TIF should be matched together.

Example: You have two image stacks in the following format:

• The stacks are Opera’s FLEX format.
• Each FLEX file contains 8 fields of view, with 3 channels at each site (DAPI, GFP, Texas Red).
• Each channel is in grayscale format.

In this case, the procedure to set up the input modules to handle these files is as follows:

• In the Images module, drag-and-drop your folders of images into the File list panel. If necessary, set your rules accordingly in order to filter out any files that are not images to be processed. In the above example, you would drag-and-drop the FLEX files into the File list panel.

• In the Metadata module, enable metadata extraction in order to obtain metadata from these files. The key step here is to obtain the necessary metadata tags to do two things:

• Distinguish the stacks from each other. This information is contained as the file itself, that is, each file represents a different stack.
• For each stack, distinguish the frames from each other. This information is usually contained in the image’s internal metadata, in contrast to the image sequence described above.

To accomplish this, do the following:

• Select “Extract from image file headers” as the metadata extraction method. In this case, CellProfiler will extract the requisite information from the metadata stored in the image headers.
• Click the “Update metadata” button. A progress bar will appear showing the time elapsed; depending on the number of files present, this step may take a while to complete.
• Click the “Update” button below the divider.
• The resulting table should show the various metadata contained in the file. In this case, the relevant information is contained in the C and Series columns. In the figure shown, the C column shows three unique values for the channels represented, numbered from 0 to 2. The Series column shows 8 values for the slices collected in each stack, numbered from 0 to 7, followed by the slices for other stacks.
• In the NamesAndTypes module, assign the channel to a name of your choice. If there are multiple channels, you will need to do this for each channel. For this example, you could do the following:

• Select “Assign images matching rules”.
• Make a new rule [Metadata][Does][Have C matching][0]
• Click the button to the right of the rule to add another set of rules underneath.
• Add the rule [Image][Is][Stack frame]. This combination tells CellProfiler not to treat the image as a single file, but rather as a series of frames.
• Name the image DAPI.
• Click the “Add another image” button to define a second image with a set of rules.
• Make a new rule [Metadata][Does][Have C matching][1]
• Click the button to the right of the rule to add another set of rules underneath.
• Add the rule [Image][Is][Stack frame].
• Name the image GFP.
• Click the “Add another image” button to define a third image with a set of rules.
• Make a new rule [Metadata][Does][Have C matching][2]
• Click the button to the right of the rule to add another set of rules underneath.
• Add the rule [Image][Is][Stack frame].
• Name the image TxRed.
• In the “Image set matching method” setting, select “Metadata”.
• Select “FileLocation” for the DAPI, GFP and TxRed channels. The FileLocation metadata tag identifies the individual stack, and selecting this parameter ensures that the channels are first matched within each stack, rather than across stacks.
• Click the button to the right to add another row, and select Series for each channel.
• Click the “Update” button below the divider to confirm that the proper image slices are listed and matched across the channels. The corresponding FileLocation and Series for each channel should now be matched to each other.
• In the Groups module, select the metadata that defines a distinct image stack. For the example above, do the following:

• Select “FileLocation” as the metadata category.
• The tables below this setting will update themselves, and you should be able to visually confirm that each of the two image stacks are defined as a group, each with 8 slices’ worth of images.

Without this step, CellProfiler would not know where one stack ends and the next one begins, and would process the slices in all stacks together as if they were constituents of only one stack.

Example: You have four Z-stacks in the following format:

• The stacks are in Zeiss’ CZI format.
• Each stack consists of a number of slices with 4 channels (DAPI, GFP, Texas Red and Cy3) at each slice.
• One stack has 9 slices, two stacks have 7 slices and the fourth has 12 slices. Even though the stacks were collected with differing numbers of slices, the pipeline to be constructed is intended to analyze all stacks in the same manner.
• Each slice is in grayscale format.

In this case, the procedure to set up the input modules to handle these this file is as follows:

• In the Images module, drag-and-drop your folders of images into the File list panel. If necessary, set your rules accordingly in order to filter out any files that are not images to be processed. In the above example, you would drag-and-drop the CZI files into the File list panel. In this case, the default “Images only” filter is sufficient to capture the necessary files.

• In the Metadata module, enable metadata extraction in order to obtain metadata from these files. The key step here is to obtain the metadata tags necessary to do two things:

• Distinguish the stacks from each other. This information is contained as the file itself, that is, each file represents a different stack.
• For each stack, distinguish the z-planes from each other, ensuring proper ordering. This information is usually contained in the image file’s internal metadata.

To accomplish this, do the following:

• Select “Extract from image file headers” as the metadata extraction method. In this case, CellProfiler will extract the requisite information from the metadata stored in the image headers.
• Click the “Update metadata” button. A progress bar will appear showing the time elapsed; depending on the number of files present, this step may take a while.
• Click the “Update” button below the divider.
• The resulting table should show the various metadata contained in the file. In this case, the relevant information is contained in the C and Z columns. The C column shows four unique values for the channels represented, numbered from 0 to 3. The Z column shows nine values for the slices represented from the first stack, numbered from 0 to 8.
• Of note in this case, for each file there is a single row summarizing this information. The sizeC column reports a value of 4 and sizeZ column shows a value of 9. You may need to scroll down the table to see this summary for the other stacks.
• In the NamesAndTypes module, assign the channel(s) to a name of your choice. If there are multiple channels, you will need to do this for each channel.

For the above example, you could do the following:

• Select “Assign images matching rules”.
• Make a new rule [Metadata][Does][Have C matching][0]
• Click the button to the right of the rule to add another set of rule options.
• Add the rule [Image][Is][Stack frame].
• Name the image DAPI.
• Click the “Add another image” button to define a second image with a set of rules.
• Make a new rule [Metadata][Does][Have C matching][1]
• Click the button to the right of the rule to add another set of rule options.
• Add the rule [Image][Is][Stack frame].
• Name the second image GFP.
• Click the “Add another image” button to define a third image with a set of rules.
• Make a new rule [Metadata][Does][Have C matching][2].
• Click the button to the right of the rule to add another set of rule options.
• Add the rule [Image][Is][Stack frame].
• Name the third image TxRed.
• Click the “Add another image” button to define a fourth image with set of rules.
• Make a new rule [Metadata][Does][Have C matching][3].
• Click the button to the right of the rule to add another set of rule options.
• Add the rule [Image][Is][Stack frame].
• Name the fourth image Cy3.
• In the “Image set matching method” setting, select “Metadata”.
• Select “FileLocation” for the DAPI,GFP,TxRed, and Cy3channels. The FileLocation identifies the individual stack, and selecting this parameter insures that the channels are matched within each stack, rather than across stacks.
• Click the button to the right to add another row, and select “Z” for each channel.
• Click “Update table” to confirm the channel matching. The corresponding FileLocation and Z for each channel should be matched to each other.
• In the Groups module, select the metadata that defines a distinct image stack. For the example above, do the following:

• Select “FileLocation” as the metadata category.
• The tables below this setting will update themselves, and you should be able to visually confirm that each of the four image stacks are defined as a group, with 9, 7, 7 and 12 slices’ worth of images.

Without this step, CellProfiler would not know where one stack ends and the next one begins, and would process the slices in all stacks together as if they were constituents of only one stack.