## New Lexicon: how to add a custom minted lexer in Overleaf

This quick post comes courtesy of LianTze Lim (an Overleaf TeXpert) and Kota Miura (a bioimage analyst).

I asked on the ImageJ forum some time ago how to add an ImageJ Macro lexer for a LaTeX document I was writing. Kota responded with this lexer for pygments. I then asked Overleaf if it was possible to add a custom lexer to an Overleaf document using the minted package. At the time this was not possible. However, I got a message from them today with a solution.

Steps to do this for your own Overleaf project:

\begin{minted}{imagejmacro.py:ImageJMacroLexer -x}
\end{minted}

Here, imagejmacro.py is the name of the custom lexer saved in your project and ImageJMacroLexer is the name of the class in that file. If you want to use another custom lexer just replace as required. I have put up a read-only Overleaf example to show it working.

Thanks to LianTze for following up with me about this and special thanks to Kota who wrote the custom lexer.

The post title comes from the LP of the same name by Paint It Black.

## Rip It Up: Grabbing movies from Twitter for use in ImageJ

Some great scientific data gets posted on Twitter. Sometimes I want to take a closer look and this post describes a strategy to do so.

Edit: I received a request to take down the 3D volume images derived from the example dataset I used in this post. I’ve edited the post below so that is now a general guide.

Grab the video

It can be a bit difficult to the grab video from Twitter. The best way I’ve found is using youtube-dl. This works for downloading video and audio from YouTube to view offline, but it also works for other embedded video content on other websites.

youtube-dl -o '%(title)s.%(ext)s' https://twitter.com/username/status/tweetID

Convert to avi

Now, mp4 is a compressed file format which cannot be read directly by FIJI/ImageJ. Conversion to avi means that the file can be loaded. I like to use another command line tool, ffmpeg for video conversions.

ffmpeg -i originalFile.mp4 -pix_fmt nv12 -f avi -vcodec rawvideo convertedFile.avi

Now we have an avi file called convertedFile.avi that we can use.

The avi can be loaded into FIJI. At this point you can analyse the video. However, in the case of the video I was interested in, the data had been pseudocolored and was now in RGB format. I wanted to look at the original data. Converting to grayscale does not give the correct representation but conversion back to grayscale is possible if you know the LUT was applied. Even if you don’t, it’s possible to take a guess at the LUT and do the conversion.

Converting RGB to original values

I found a nice gist that does the conversion for a single image. I just modified this code to work for a stack. It requires the LUT to be displayed vertically in a window called LUT. Caution: this code runs very slowly because every pixel in every slice needs to be recalculated and ImageJ is slow… I took a guess that mpl-inferno was used (I don’t think is exactly right but it worked well enough). You can display the built-in LUTs in FIJI using Color > Display LUTs… and from there you can make the LUT window which the macro uses for the calculation. The macro to convert stacks to grayscale using the LUT is here.

I had a nice grayscale version of the data (inverted because I wanted to look at the volume). This let me see how the layers in the original video add together to make the full structure. I used ClearVolume which can be installed via Update Site in FIJI. I just made a quick video to show it in action (see below). You’ll have to take my word for it (video removed).

So extracting scientific data from Twitter or another online source is pretty straightforward. The extra complication was getting rid of the pseudocoloring, but once this was done, something very close to the original data was available.  Nonetheless this workflow is a fun way to take a closer look at some of the cool movies that people post on Twitter. I hope you find it useful.

The post title comes from “Rip It Up” by Orange Juice. A popular title in my library with versions from several different artists. I was thinking what is described is similar to ripping video content.

## Adventures in Code III: the quantixed ImageJ Update site

We have some macros for ImageJ/FIJI for making figures and blind analysis which could be useful to others.

I made an ImageJ Update Site so that the latest versions can be pushed out to the people in the lab, but this also gives the opportunity to share our code with the world. Feel free to add the quantixed ImageJ update site to your ImageJ or FIJI installation. Details of how to do that are here.

The code is maintained in this GitHub repo, which has a walkthrough for figure-making in the README. So, if you’d like to make figures the quantixed way, adding ROIs and zooms, then feel free to give this code a try. Please raise any issues there or get in touch some other way.

Disclaimer: this code is under development. I offer no guarantees to its usefulness. I am not responsible for data loss or injury that may result from its use!

Update @ 10:35 2016-12-20 I should point out that other projects already exist to make figures (MagicMontage, FigureJScientiFig). These projects are fine but they didn’t do what I wanted, so I made my own.

## Blind To The Truth

Molecular Biology of The Cell, the official journal of the American Society for Cell Biology, recently joined a number of other periodicals in issuing guidelines for manuscripts, concerning statistics and reproducibility. I discussed these guidelines with the lab and we felt that there are two areas where we can improve:

• blind analysis
• power calculations

A post about power analysis is brewing, this post is about a solution for blind analysis.

For anyone that doesn’t know, blind analysis refers to: the person doing the analysis being blind to (not knowing) the experimental conditions. It is a way of reducing bias, intentional or otherwise, of analysis of experimental data. Most of our analysis workflows are blinded, because a computer does the analysis in an automated way so there is no way of a human biasing the result. Typically, a bunch of movies are captured, fed into a program using identical settings, and the answer gets spat out. Nothing gets excluded, or the exclusion criteria are agreed beforehand. Whether the human operating the computer is blind or not to the experimental conditions doesn’t matter.

For analysis that has a manual component we do not normally blind the analyser. Instead we look for ways to make the analysis free of bias. An example is using a non-experimental channel in the microscope image to locate a cellular structure. This means the analysis is done without “seeing” any kind of “result”, which might bias the analysis.

Sometimes, we do analysis which is almost completely manual and this is where we can improve by using blinding. Two objections raised to blinding are practical ones:

• it is difficult/slow to get someone else to do the analysis of your data (we’ve tried it and it doesn’t work well!)
• the analyser “knows” the result anyway, in the case of conditions where there is a strong effect

There’s not much we can do about the second one. But the solution to the first is to enable people to blindly analyse their own data if it is needed.

I wrote* a macro in ImageJ called BlindAnalysis.ijm which renames the files in a random fashion** and makes tsv log of the associations. The analyser can simply analyse blind_0001.tif, blind_0002.tif and then reassociate the results to the real files using this tsv.

The picture shows the macro in action. A folder containing 10 TIFFs is processed into a new folder called BLIND. The files are stripped of labels (look at the original TIFF, left and the blind version, right) and saved with blinded names. The log file keeps track of associations.

I hope this simple macro is useful to others. Feedback welcome either on this post or on GitHub.

* actually, I found an old macro on the web called Shuffler written by Christophe Leterrier. This needed a bit of editing and also had several options that weren’t needed, so it was more deleting than writing.

** it also strips out the label of the file. If you only rename files, the label remains in ImageJ so the analyser is not blind to the condition. Originally I was working on a bash script to do the renaming, but when I realised that I needed to strip out the labels, I needed to find an all-ImageJ solution.

Edit @ 2016-10-11T06:05:48.422Z I have updated the macro with the help of some useful suggestions.

The post title is taken from “Blind To The Truth” a 22 second-long track from Napalm Death’s 2nd LP ‘From Enslavement To Obliteration.

## Everything In Its Right Place

Something that has driven me nuts for a while is the bug in FIJI/ImageJ when making montages of image stacks. This post is about a solution to this problem.

What’s a montage?

You have a stack of images and you want to array them in m rows by n columns. This is useful for showing a gallery of each frame in a movie or to separate the channels in a multichannel image.

What’s the bug/feature in ImageJ?

If you select Image>Stacks>Make Montage… you can specify how you want to layout your montage. You can specify a “border” for this. Let’s say we have a stack of 12 images that are 300 x 300 pixels. Let’s arrange them into 3 rows and 4 columns with 0 border.

So far so good. We have an image that is 1200 x 900. But it looks a bit rubbish, we need some grouting (white pixel space between the images). We don’t need a border, but let’s ignore that for the moment. So the only way to do this in ImageJ is to specify a border of 8 pixels.

Looks a lot better. Ok there’s a border around the outside, which is no use, but it looks good. But wait a minute! Check out the size of the image (1204 x 904). This is only 4 pixels bigger in x and y, yet we added all that grouting, what’s going on?

The montage is not pixel perfect.

So the first image is not 300 x 300 any more. It is 288 x 288. Hmmm, maybe we can live with losing some data… but what’s this?

The next image in the row is not even square! It’s 292 x 288. How much this annoys you will depend on how much you like things being correct… The way I see it, this is science, if we don’t look after the details, who will? If I start with 300 x 300 images, it’s not too much to ask to end up with 300 x 300 images, is it? I needed to fix this.

Solutions

I searched for a while for a solution. It had clearly bothered other people in the past, but I guess people just found their own workaround.

ImageJ solution for multichannel array

So for a multichannel image, where the grayscale images are arrayed next to the merge, I wrote something in ImageJ to handle this. These macros are available here. There is a macro for doing the separation and arraying. Then there is a macro to combine these into a bigger figure.

Igor solution

For the exact case described above, where large stacks need to be tiled out into and m x n array, I have to admit I struggled to write something for ImageJ and instead wrote something for IgorPRO. Specifying 3 rows, 4 columns and a grout of 8 pixels gives the correct TIFF 1224 x 916, with each frame showing in full and square. The code is available here, it works for 8 bit greyscale and RGB images.

I might update the code at some point to make sure it can handle all data types and to allow labelling and adding of a scale bar etc.

The post title is taken from “Everything In Its Right Place” by Radiohead from album Kid A.

## Tips from the blog VII: Recolour Z-stack and Save Projection

I’m putting this up here in case it is useful for somebody.

We capture Z-stacks on a Perkin Elmer Spinning Disk microscope system. I wanted to turn each stack into a single image so that we could quickly compare them. This simple macro does the job.

1. We import the images straight from the *.mvd2 library using the wonderful BioFormats import tool. We open all files as composite hyperstacks.
2. This Macro is then run (works on all of the open images).
3. It finds the mid-Z point of the stack and sets the brightness/contrast for each channel to Auto.
4. It then recolours the channels to the right colours (we capture DAPI then GFP then mCherry, but the import colours them red, green and blue, respectively).
5. Then it makes a z-projection and saves the file in a directory that you specify at the start.
6. It closes the projection and the z-stack.

You can then open up all of the projections, tile them and take a look at what was going on in the experiment.


setBatchMode(true);
imgArray = newArray(nImages);
dir1 = getDirectory("Choose Destination Directory ");
for (i=0; i<nImages; i++) {
selectImage(i+1);
imgArray[i] = getImageID();
}
for (i=0; i< imgArray.length; i++) {
selectImage(imgArray[i]);
id = getImageID();
win = getTitle();
getDimensions(w, h, c, nFrames, dummy);
Stack.setSlice(round(nFrames/2));
run("Enhance Contrast", "saturated=0.35");
run("Next Slice [>]");
run("Enhance Contrast", "saturated=0.35");
run("Next Slice [>]");
run("Enhance Contrast", "saturated=0.35");
Stack.setChannel(1)
run("Blue");
Stack.setChannel(2)
run("Green");
Stack.setChannel(3)
run("Red");
run("Z Project...", "projection=[Max Intensity]");
saveAs("TIFF", dir1+win);
close();
selectImage(id);
close();
}



Sorry for lack of commenting.

Tips from the blog is a series and gets its name from a track from Black Sunday by Cypress Hill.