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.

imgArray = newArray(nImages);
dir1 = getDirectory("Choose Destination Directory ");
for (i=0; i<nImages; i++) {
	imgArray[i] = getImageID();
for (i=0; i< imgArray.length; i++) {
	id = getImageID();
	win = getTitle();
	getDimensions(w, h, c, nFrames, dummy);
	run("Enhance Contrast", "saturated=0.35");
	run("Next Slice [>]");
	run("Enhance Contrast", "saturated=0.35");
	run("Next Slice [>]");
	run("Enhance Contrast", "saturated=0.35");
	run("Z Project...", "projection=[Max Intensity]");
	saveAs("TIFF", dir1+win);

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.

Creep Diets: Fewer papers published at JCB

JCBdietA couple of years ago, a colleague sent me this picture* to say “who put J Cell Biol on a diet?”. I joked that maybe they publish too many autophagy papers and didn’t think much more of it.

Recently, Ron Vale put up this very interesting piece on bioRxiv discussing what it takes to publish a paper in the field of cell biology these days. In the main, he questions whether this is now out of reach of many trainees in our labs. It raises some great points and I recommend reading it.

One (of many) interesting stats in the article is that J Cell Biol now publishes fewer papers than it used to. Which made me think back to the photo and wonder why there has been a decline. Elsewhere, Vale notes that a cell biology paper now contains >2 the amount of data than papers of yesteryear. I’ve also written before about the creeping increase in the number of authors per paper at J Cell Biol and (more so) at Cell. Publication in Science is something of an arms race and his point is really that the amount of data, the time taken, the effort/people involved has got to an untenable level.

The data in the preprint is a bit limited as he only looks at two snapshots in time – because he looks at two cohorts of students at UCSF. So I thought I’d look at the decrease in JCB papers over time – did it really fall off? by how much? when did it start?.


Getting the data is straightforward. In fact, PubMed will give you a csv of frequency of papers for a given search term (it even shows you a snapshot in the main search window). I wanted a bit more control, so I exported the records for JCB and NCB. I filtered out interviews and commentary as best as I could and plotted out the records as two histograms using a bin width of 6 months. It’s pretty clear that J Cell Biol is indeed publishing fewer papers now than it used to. It looks like the trend started around 2002, possibly accelerating in the last 5 years (the photo agrees with this). The six month output at JCB in 2015 is similar to what it was in 1975!

In the comments section of the preprint, there is a bit of discussion of why this may be. Overall, there are more and more papers being published every year. There’s no reason to think that the number of cell biology papers has remained static or fallen. So if J Cell Biol have not taken a decision to limit the number of papers, why is there a decline? One commenter suggests  Nature Cell Biology has “taken” some of these papers. So I plotted those numbers out too. The number of papers at NCB is capped and has been constant since the launch of the journal. It does look like NCB could be responsible, but it’s a complex question. Personally, I think it’s unlikely. When NCB was launched this marked a period of expansion in the number of scientific journals and it’s likely that the increase in number of venues that a paper can go to (rather than the creation of NCB per se) has affected publication at JCB. One simple cause could be financial, i.e. the page number being limited by RUP. If this is true, why not move the journal online? There’s so many datasets and movies in papers these days that it barely makes sense to print JCB any more.

I love reading papers in JCB. They are sufficiently detailed so that you know what’s going on. They’re definitely on Cell Biology, not some tangential area of molecular biology. The Editors are active cell biologists and it has had a long history of publishing some truly landmark discoveries in our field. For these reasons, I’m sad that there are fewer JCB papers these days. If it’s an editorial decision to try to make the journal more exclusive, this is even more regrettable. I wonder if the Editors feel that they just don’t get enough high quality papers. If this is the case, then maybe the expectations for what a paper “should be” need to be brought back in line with reality. Which is one of the points that Ron Vale is making in his article.

* I cropped the picture to remove some identifying things on the bookshelf.

Update @ 07:07 17/7/15: Rebecca Alvinia from JCB had left a comment on Ron Vale’s piece on bioRxiv to say that JCB are not purposely limiting the number of papers. Fillip Port then asked why JCB does not take preprints. Rebecca has now replied saying that following a change of policy, J Cell Biol and the other RUP journals will take preprinted papers. This is great news!

Creep Diets is the title track from the second album by the oddly named Fudge Tunnel, released on Earache Records in 1993

Tips from the blog VI: doc to pdf

doctopdfA while back I made this little Automator script to convert Microsoft Word doc and docx files to PDF. It’s useful for when you are sent a bunch of Word files for committee work. Opening PDFs in Preview is nice and hassle-free. Struggling with Word is not.

It’s not my own work, I just put it together after googling around a bit. I’ll put it here for anyone to use.

To get it working:

  1. Open Automator. Choose Template Service and you need to check: Service receives selected files and folders in
  2. Set the actions: Get Folder content and Run AppleScript (this is where the script goes)
  3. Now Save the workflow. Suggested name Doc to PDF.

Now to run it:

  1. Select the doc/docx file(s) in the Finder window.
  2. Right-click and look for the service in the contextual menu. This should be down the bottom near “Reveal in Finder”.
  3. Run it.

If you want to put it onto another computer. Go to ~/Library/Services and you will find the saved workflow there. Just copy to the same location on your other computer.

Known bug: Word has to be open for the script to run. It also doesn’t shut down Word when it’s finished.

Here is the code.

property theList : {"doc", "docx"}

on run {input, parameters}
          set output to {}
          tell application "Microsoft Word" to set theOldDefaultPath to get default file path file path type documents path
          repeat with x in input
                              set theDoc to contents of x
                              tell application "Finder"
                                        set theFilePath to container of theDoc as text

                                        set ext to name extension of theDoc
                                        if ext is in theList then
                                                  set theName to name of theDoc
                                                  copy length of theName to l
                                                  copy length of ext to exl

                                                  set n to l - exl - 1
                                                  copy characters 1 through n of theName as string to theFilename

                                                  set theFilename to theFilename &amp; ".pdf"

                                                  tell application "Microsoft Word"
  set default file path file path type documents path path theFilePath
                                                            open theDoc
                                                            set theActiveDoc to the active document
  save as theActiveDoc file format format PDF file name theFilename
                                                            copy (POSIX path of (theFilePath &amp; theFilename as string)) to end of output
  close theActiveDoc
                                                  end tell
                                        end if
                              end tell
                    end try
          end repeat
          tell application "Microsoft Word" to set default file path file path type documents path path theOldDefaultPath

          return output
end run


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

Pull Together: our new paper on “The Mesh”

We have a new paper out! You can access it here.

Title of the paper: The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle

bundle1What’s it about? When a cell divides, the two new cells need to get the right number of chromosomes. If this process goes wrong, it is a disaster which may lead to disease e.g. cancer. The cell shares the chromosomes using a “mitotic spindle”. This is a tiny machine made of microtubules and other proteins. We have found that the microtubules are held together by something called “the mesh”. This is a weblike structure which connects the microtubules and gives them structural support.

Does this have anything to do with cancer? Some human cancer cells have high levels of  proteins called TACC3 and Aurora A kinase. We know that TACC3 is changed by Aurora A kinase. This changed form of TACC3 is part of the mesh. In our paper we mimic the cancer condition by increasing TACC3 levels. The mesh changes and the microtubules become wonky. This causes problems for dividing cells. It might be possible to target TACC3 using drugs to treat certain types of cancer, but this is a long way in the future.

Who did the work? Faye Nixon, a PhD student in the lab did most of the work. She used a method to look at mitotic spindles in 3D to study the mesh. My lab actually discovered the mesh by accident. A previous student, Dan Booth – back in 2011 – was looking at mitotic spindles to try and get 3D electron microscopy (tomography) working in the lab. Tomography works just like a CAT scan in a hospital, but on a much smaller scale. The mesh is found in the gaps between microtubules that are 25 nanometre wide (1 nanometre is 1 billionth of a metre), this is about 3,000 times smaller than a human hair, so it is very small! It was Dan who found the mesh and gave it the name. Other people in the lab did some really nice work which helped us to understand how the mesh works in dividing cells. Cristina Gutiérrez-Caballero did some experiments using a different type of microscope and Fiona Hood contributed some test tube experiments. Ian Prior at University of Liverpool, co-supervises Faye and helped with electron microscopy.

Have you discovered a new structure in cells? Yes and No. All cell biologists dream of finding a new structure in cells. It’s so unlikely though. Scientists have been looking at cells since the 17th Century and so the chances of seeing something that no-one has seen before are very small. In the 1970s, “inter-microtubule bridges” in the mitotic spindle were described using 2D electron microscopy. What we have done is to look at these structures in 3D for the first time and find that they are a network rather than individual connectors.

The work was funded by Cancer Research UK and North West Cancer Research Fund.


Nixon, F.M., Gutiérrez-Caballero, C., Hood, F.E., Booth, D.G., Prior, I.A. & Royle, S.J. (2015) The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle eLife, doi: 10.7554/eLife.07635

This post is written in plain English to try to describe what is in the paper. I’m planning on writing a more technical post on some of the spatial statistics we developed as part of this paper.

The post title is from “Pull Together” a track from Shack’s H.M.S. Fable album.

Green is the Colour: mNeonGreen spectra

I was searching for the excitation and emission spectra for mNeonGreen. I was able to find an image, but no values for the spectra. Here is an approximation of the spectra (xlsx format, still haven’t figured out csv for wordpress).

I got these values using IgorThief.ipf a very handy tool that allows the extraction of XY coordinates from a bitmapped plot (below).

mNeonGreen is available from Allele Biotechnologies.

Here is a great site for comparing fluorescent protein properties.

Edit @ 06:54 4/7/14: A web-based data thief was suggested by @dozenoaks

The post title is taken from “Green is the Colour” from the Pink Floyd LP “More”