imaging biology notes

A mitotic wave

A mitotic wave traveling through an early #Drosophila #embryo #FlyFriday

3D HisGap cleavage cover
Early syncytial embryo of the fly Drosophila melanogaster. Nuclei (blue) are dividing in a wave from posterior to anterior. Membrane components (white) are already organized around the nuclei. The image is a frame from a timelapse acquired under lightsheet microscopy and rendered in 3D.
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. 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.

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.

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

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

And here are the same two movies now synchronized by embryonic stage:

The same two embryos, now synchronized.

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. Here are the instructions step-by-step:

  1. Open both image stacks in ImageJ.
  2. Adjust 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.

I’ve 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.

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?

biology imaging notes

Chubby ribbon worm

A chubby ribbon worm juvenile #Nemertean #WormWednesday

Chubby ribbon worm
Juvenile specimen of the nemertean Lineus ruber under wide field fluorescence microscopy. Magenta: Nuclei; Green: F-actin.

biology imaging notes

Larva of a lamp shell

Larva of a lamp shell, also known as brachiopod.
Terebratalia transversa (Sowerby, 1846)
Oil on canvas
167.95 µm × 167.95 µm
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.

imaging biology notes

The surface of a brachiopod embryo

Brachiopod embryo showing its surface and blastopore.
Embryo of the brachiopod Novocrania anomala at the gastrula stage showing its outer surface and the blastopore at the bottom. Cell membranes were stained (F-actin) and the original image stack was converted to a 3D animation using Fiji/ImageJ.
imaging notes

A typical lightsheet microscopy session

Here is how a typical session of imaging embryos under Lightsheet Microscopy goes. A glimpse into my day-to-day work :)

Assemble the incubation chamber:

Collect and mount the embryos:

Typical lightsheet microscopy session.

Acquire a short timelapse from multiple angles:

Transfer (lots of) data for image processing ;)

File transfer
biology imaging notes

The pelagosphera larva of Sipuncula

The pelagosphera larva of #Sipuncula. Photo by Alvaro Migotto via @cifonauta

Pelagosphera larva of Sipuncula
biology imaging notes

A beautiful nemertean

The ribbon worm Tubulanus #WormWednesday #Nemertea

The nemertean Tubulanus.
Adult specimen of the nemertean Tubulanus annulatus collected in Norway.