Combine images into a multichannel image or stack

Overview

 

This mode converts single-channel images to multichannel images or image stacks. Both 2D and 3D images/image stacks can be combined into multichannel data.

See the list of compatible file formats.

Example: Convert single-channel red, green, and blue 2D tiff images into a 2D multichannel image in JPEG 2000 and/or OME TIFF format, or convert single-channel red and green 3D image stacks into a 3D multichannel stack.

The procedure includes 4 steps (click on steps to jump to procedure details):

  1. Choose conversion settings
  2. Select source files and destination directory. Choose the type of output and identify
  3. View and/or edit metadata
  4. Click to convert the files

Procedure

  1. Click the button, Combine images into a multichannel image or stack to choose file-conversion settings.

    1. Save Format: Choose the format for your output image file:

      • JPEG 2000: file extension ".jpx" (learn more about the format)
      • OME TIFF: file extension ".tif" (learn more about the format)
      • Both: saves as both file types

    2. JPEG 2000 Compression: If you chose JPEG 2000 or Both, choose the compression settings for the JPEG 2000 image; file conversion to this format can be compressed (lossy) or uncompressed (lossless).

      • It is recommended to select lossless compression when converting images approximately 1 GB or less in size, or when converting a JPEG.
      • If you choose Lossy compression, indicate the compression ratio desired.

    3. Advanced: Other available options

      • Skip .jp2 (JPEG 2000) files
      • Force incremental saving
      • Require metadata entry before saving: this option is selected by default

  2. Click Next to select source file(s), indicate the output destination, and specify how color channel and stack position is shown in file names.

    Images selected or dragged and dropped together will be combined into one multi-channel image or 3D stack. Multiple images or stacks can be created by selecting or dragging and dropping each series of images separately.

    1. Select the image(s) to combine via drag and drop or navigating and selecting:

      • Drag and drop: select images as a group and drag into the MicroFile+ window together.

      • Click the Load files.... button, navigate to the location of the file(s) you want to convert, select the group of images to combine, and press Open.

    2. The Multichannel Options window opens and asks if you want to combine 2D images into a 3D stack.

      • Click the radio button to answer Yes or No.

      • Click the Load button.

    3. If you answered yes to the previous question (i.e., you want to combine 2D images into a 3D stack) the Multichannel Options window remains open so that you can provide requested information on file naming conventions:

      1. Select output: the Yes radio button is selected as per your previous answer to combine 2D images into a 3D stack. (Note that you can change this if needed.)

      2. Assemble Images: indicate how the color channel and image plane are captured in the file names. MicroFile+ software uses this information to properly merge and stack images. Choose one of the two methods for doing this:

      • Dimension Ordering uses the file names as is. To use this option:

        • Enter the number of color channels in your images

        • Review the table under 3. Confirm Assembly. If the color channel and image planes were switched, click the check box for Order channels before planes and review the assignations again.

      • Pattern Matching asks you to indicate how file names show color channel numbers and image planes.

        • The first file name in the list is displayed in the Pattern field. Follow the onscreen instructions to indicate how the color channel and image plane are captured in the file names by replacing them with an asterisk symbol (*) and a hashtag symbol (#), respectively.

          As you do this, you will see the table under 3. Confirm Assembly populate based on your input.

        • If any of the file names include unique text that is not present in all file names, replace it with a question mark (?) in the text box.

      3. Confirm Assembly: Review the table to verify that all files are recognized as the appropriate color channel and image plane; edit the values in the table if necessary.

      4. Other Notes:

      • The file(s) will appear as groups in the MicroFile+ window.
      • To remove a file from the conversion window, select the file and press Remove Selected.

    4. Click the Load button.

    5. Repeat the file selection process to combine multiple sets of files.

    6. Destination for image stack(s) after conversion:

      • By default, image stack(s) are saved to the same location as the original(s).

      • To select a different location for all stacks in the window, click the Select Output... button, navigate to the destination folder, and click Select Folder.

      • To select a different location for individual stacks in the window, click the Save button next to the file name, navigate to the destination folder, click Open, and click Save.

  3. Click Show Metadata to view/edit metadata.

    • The Show Metadata button is in the lower left-hand corner of the window.
    • Click to select a file stack in the conversion window and view the metadata for that stack in the Metadata Generation panel on the right.

    • The status of metadata completion is indicated next to each stack:

      • Green check mark indicates that critical metadata is sufficient.
      • Red X indicates that critical metadata is missing. Click the file name and the critical metadata fields are highlighted in yellow. Pink boxes indicate fields that lack information. Double-click to enter metadata.

    Learn more about entering/modifying metadata here.

    See the Metadata Glossary for more information on each metadata field

  4. Click Convert to begin the conversion process.

    • If there is a file with the same name in the output directory, the "File Already Exists" dialog opens. Click yes to proceed and “converted” will be added to the end of the file name to distinguish the new file conversion process.