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imaging articles code

ImageJ macro to synchronize and combine image stacks

The embryos I study rarely develop in perfect synchrony. That means that when I film them under the microscope some embryos will be younger—or older—than others.

ImageJ macro with Drosophila embryo
Using an ImageJ macro to help me analyze movies of Drosophila embryos.

For this reason, I often need to synchronize the recordings to make sure they all begin at the same embryonic stage. When the movies are synchronized I can combine them side-by-side, and it becomes much easier to compare and spot differences between two embryos.

ImageJ macros save time

Combining movies in Fiji/ImageJ is straightforward using the Combine... command. But synchronizing is way harder. It depends on human classification and involves some calculations and stack juggling that can (and will) become tedious.

To help me out, I wrote a small ImageJ macro available here: SyncAndCombineStacks.ijm. Follow below to see how it works.

Combined movies without syncing

That’s what unsynchronized movies look like. I combined them fresh off the microscope without any synchronization:

Two embryos of the fruit fly Drosophila melanogaster. Both were acquired in the same microscopy session. The top embryo is older than the bottom embryo.

Combined movies after syncing

Here are the same two movies now synchronized by the embryonic stage:

The same two embryos are now synchronized.

How it works

The macro performs the hard work. It calculates how many frames to trim from each stack. Then it duplicates the selected range of frames common to both stacks. Finally, it combines the synchronized recordings into a single image stack. All you need to do is to select the corresponding frames between the two stacks.

Step-by-step instructions

Here are the instructions step-by-step:

  1. Open both image stacks in ImageJ.
  2. Adjust the contrast if needed (before running the macro).
  3. Select a reference frame in the top stack (e.g. stage easy to recognize).
  4. Select the correspondent frame in the bottom stack.
  5. Run the macro and fill in the dialog parameters.
  6. Click OK, wait a few seconds, and check if the synchronization is good. Otherwise, re-run with different parameters.

Screencast

I’ve also recorded a small screencast:

Note! The macro does not touch the original stacks, but it outputs an RGB Color stack. There are a couple of reasons for that. Converting to RGB avoids contrast issues when the stacks have different pixel intensities. It also prevents quirks in video players that can’t handle 16-bit movies. But if you need to perform image analyses on the final stack, remove this option. I may add a checkbox for that in the future.

Categories
biology imaging notes

The blastopore of bryozoan embryos

This is a bryozoan embryo exhibiting its blastopore. These animals are discreet but ubiquitous in oceans and lakes all over the world.

Bryozoan embryo during gastrulation revealing its blastopore.
Embryo of the bryozoan Membranipora membranacea under confocal microscopy.

What we see is the DNA inside the nucleus of the cells of the embryo. The color gradient indicates if the nuclei are closer (yellow) or further away (purple) from the microscope camera.

The embryonic cells are arranged in a circle and form a central opening that we call the blastopore. This opening, in bryozoans, will become the mouth of the animal after the embryo develops.

You can follow the process on video or learn more details in the paper.

What about our mouth, where does it come from?

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articles code

Convert video to animated GIF

Something that I began doing more often is converting videos of developing embryos or marine invertebrates to animated GIFs. But how to do this conversion without affecting the quality of the video?

A jellyfish moving its tentacles. Source: Cifonauta.

Some time ago I found this guide to convert videos to high-quality animated GIFs using the tool FFmpeg. The trick is to generate a color palette based on the original video to improve the color quality of the GIF. Based on this guide I created a small bash script to make my life easier and perhaps yours too ;)

Check it in https://github.com/nelas/gif.sh

Categories
biology imaging notes

Fruit fly embryo under lightsheet microscopy

A short video that I made about the embryonic development of the likeable Drosophila, also known as fruit fly or vinegar fly, won an honorable mention in the Small World in Motion.

A single embryo imaged from four different angles.

The details on the techniques I used and the video on its full resolution are available for download and re-use on the Wikimedia Commons.