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4d Tools

Overview

4d Tools is a set of macros/plugin for (1) import of legacy 4d datasets as movies (.MOV, .MP4, .AVI); (2) simplified import of Imaris .IMS and Micro-manager 1.4/2.0 datasets into Fiji; and (3) experimental playback of maximum-intensity projections on the fly as a crude attempt to recaptiulate legacy Perkin-Elmer UltraView software.

Rationale

  1. Import of legacy datasets is useful for many long-established C. elegans laboratories, for example, which have large numbers of legacy 4d DIC datasets as movies in xytc format. For this vintage approach, see, for example, Thomas, C., DeVries, P., Hardin, J., and White, J. (1996). Four-dimensional imaging: computer visualization of 3D movements in living specimens. Science 273, 603-607.

  2. Micro-Manager has an integrated playback mechanisms built on top of ImageJ, and a free multi-platform Imaris player is avaialble, but in some cases direct import into Fiji is useful, and these macros simplify this process for routine work.

  3. Oddly, there doesn’t seem to be a stratighforward way to display a “range” of Z slices as a maximium intensity projection in ImageJ/Fiji. Old Perkin-Elmer Ultraview software had this functionality.

Installation in Fiji

Add the 4d-Tools update site using the Fiji update sites manager. After updating, five new menu itms will be aqdded to Fiji:

4d Import options

A “4d” submenu will be added to the “Import” menu in Fiji. Four items will be avaialble in this submenu:

  1. Classic 4d AVI to Hyperstack - Imports a 4d AVI video as a Virtual Hyperstack. It looks for a plain text file with the same root name as video. If none is found the macro will ask for the number of focal planes.
  2. Classic 4d Movie to Hyperstack - Imports a 4d Quicktime .MOV or an MPEG4 .MP4 video as a 4d Virtual Hyperstack using FFMpeg. It looks for a plain text file with the same root name as the video. If none is found the macro will ask for the number of focal planes.
  3. Imaris to Hyperstack - Imports the highest- resolution dataset in an Imaris .IMS file (a variant of the HDF5 format) as a Virtual Hyperstack using the BioFormats Importer built into Fiji.
  4. MM Images to Hyperstack - Imports a Micro-Manager 1.4 or 2.0 dataset as a Virtual Hyperstack, using the metadata in the dataset.

Hyperstack Projector

A new “Hyperstack Projector” item will be added to the “Image -> Hyperstacks” submenu. Running this plugin will attempt to recaptiulate the functionality of the old Perkin-Elmer Ultraview software, which performed a maximum-intensity projection on a specified range of images on the fly. The plugin assumes a 4d Hyperstack is open, and temporarily replaces the stack window with a custom window that allows specificaiton of the number of focal planes to be projected. CLosing the window restores the original stack window.

NOTE: This plugin is extremely experimental. The code in the installed .JAR file can likely be massively improved.

Usage

Classic 4d AVI to Hyperstack

  1. Select menu “Import->4d-> Classic 4d AVI to Hyperstack”.
  2. Specify the video file.
  3. If no text file with focal plane information is found, specify the number of focal planes.
  4. The dataset will be loaded as a Virtual Hyperstack.

Classic 4d Movie to Hyperstack

  1. Select menu “Import->4d-> Classic 4d Movie to Hyperstack”.
  2. Specify the video file.
  3. If no text file with focal plane information is found, specify the number of focal planes.
  4. The dataset will be loaded as a Virtual Hyperstack.
  5. A window with the FFMpeg import data from the FFmpegVideoImportExport plugin should be visible.

Imaris to Hyperstack

  1. Select menu “Import->4d-> Imaris to Hyperstack”.
  2. Specify the data file.
  3. The dataset will be loaded as a Virtual Hyperstack.

MM Images to Hyperstack

  1. Select menu “Import->4d-> MM Images to Hyperstack”.
  2. Specify the data directory.
  3. The dataset will be loaded as a Virtual Hyperstack.

Hyperstack Projector

  1. With a 4d Hyperstack in the frontmost window, select menu “Import->Hyperstacks-> Hyperstack Projector”.
  2. The Hyperstack window will be replaced with a custom window.
  3. Enter the number of focal planes to be projected centered on the current focal plane in the “d” text field.
  4. A running projection will be displayed centered on the current z plane and time point.
  5. Navigate up and down in space using the “z” slider and forward and backward in time using the “>” slider. Clicking the “v” button will play the movie, which will loop if the “loop” checkbox is selected.

Additional information and support

Sample data can be downloaded from the Hardin Lab microscopy page