Greetings, Earthlings: just emerging from my self-imposed laboratory exile for a quick update. I do still exist, and my radio silence can be explained by the fact that I’m in the home stretch of my resubmission. For those of you who’ve lost the plot, I’m referring to my big screen paper, which I sent to an excellent cell biology journal some time ago. After three largely positive referees, and a heart-warmingly encouraging editorial letter about returning paper with just One More Experiment appended, I am almost at the end.
As explained previously, just to cover my bases, the Experiment soon morphed into several, a Gold Standard and a few Backups. Much to my bewilderment, the Experiments, both Gold and otherwise, have exceeded my expectations and are, quite frankly, eating out of my hand. I’m currently in the process of doing the final Gold replicate, putting the finishing touches to the manuscript, and supervising a lovely summer student who fell onto my head at probably the worst possible time, but – what the heck. She’s smart and enthusiastic and has managed to master the art of opening a 15 mL Falcon tube with a finger and thumb and yet somehow managing to keep the cap a safe distance away from the tube neck during extreme tissue culture. Who could ask for more?
Arcane arts
In sum, this is not going to be the insightful blog post of your dreams. Instead, I leave you with the following tantalizing picture as a place-holder – one of the images from one of my backup experiments, expressing a construct I received recently.
Isn’t she a beaut?
Gorgeous.
“..encouraging editorial letter about returning paper with just One More Experiment appended, I am almost at the end.”
That “almost” seem like forever…at least for me it does!
A paper I first submitted a year ago is now at that stage. Not with the initial journal I had hoped would accept it (or the second – I am a bit too high!), but still respectable enough. Unfortunately I have to rely on a former lab mate to do the extra experimental work required, but luckily it is her inital contribution that needs the extra bit of data.
Good luck!
You’re right, Dr G – even my laundry list of minor text changes seems to stretch out forever. I keep wishing I could snap my fingers and have it magically done for me. Good luck with your own!
That’s a lovely picture, that is.
I’m guessing DAPI or Hoechst for the blue nuclei and rhodamine phalloidin for red actin filaments… but I imagine we’ll have to wait for your paper to find out what the green staining is?
One Q: is this confocal with ‘colour reconstruction’ in the computer, or one of those three-colour filter cubes and a colour camera on a light microscope? I’m sure I should be able to tell.
Hi Austin – you’re right about the DAPI and the TRITC-Phalloidin, No great secret about the green guy, which is a construct bearing the PBD of human Pak fused to YFP – it will bind exclusively to Rac1 in its active, GTP-bound state, so can be used as a probe for active Rac. It’s taken on a Leica SP5 confocal and I’ve used ImageJ to false-color and merge the channels.
So how did you solve the concentric circles cell incubator vibration problem?
Just a point, false coulouring with reds and greens is a right pain in the bottom to we colourblind types. My former colleague at the Institute of Ophthalmology Tim Levine wrote an article in the journal Traffic about how to make data more accessible to the significant minority of us who are colour vision different to the norm. After all, we may be your referees.
http://eprints.ucl.ac.uk/15124/1/15124.pdf
I do EM. It’s all black and white!
Lovely photo. Might I suggest that it could form the design concept for Princess Beatrice’s next wedding hat?
Ok, ok, I’m on my way out.
The PBD of human Pak fused to YFP. Yes. Just as I thought.
Oh, nice article – thanks for the link, I’m going to circulate this at work.
At a lab meeting last week, someone presented data represented by red circles (different sizes and colour intensities) on a green background. The figure was taken from a manuscript that’s currently under review. I pointed out (privately, after the presentation) that a colour-blind reader wouldn’t be able to interpret the figure, and the person said they’d literally never considered that before!
Fear not – that photo is not yet publication quality. I need to look for co-localization so, for the purposes of rough and ready analysis, red+green=yellow is the only thing that my poor brain can cope with. I just don’t cope as well with magenta at this stage.
I should also mention that whenever I show colored cells like this, I always present the relevant channels in greyscale alongside.
I thought that hat had been designed by Coiture de Cthulhu.
Good question. Dr Rohn, your public clamors to know.
Apparently they were doing some building works which have now ceased. I haven’t seen the problem for a few days now, but am still being vigilant!
There are some webpages you can use to see what a graphic would look like to someone who is colourblind. I’ve used this one before, but it’s ignoring me at the moment, so I don’t know if it’s still active.
I am occasionally alerted to this problem by colourblind referees of manuscripts who have trouble interpreting some colour figures.
I would expect nothing less of you 😉
What analysis technique are people using to show colocalization these days?
Speaking as a grumpy semi-retired fluorescence microscopist, I remember that a decade or so back I used to be chewing the carpet when people just showed yellow pixels in the red/green pictures, waved their hands and said ‘colocalization’. You used to see it in papers too. I guess these days there must be ImageJ plugins to do it properly w intensity correlation analysis, or something.
Also curious to know if everyone uses the same technique – guess Steve Caplan must do shedloads of fluorescence colocalization stuff too.
No idea. I’m just idly, not rigorously curious – wouldn’t publish co-localization just based on yellowness – though people still do this. Plus the interaction I’m looking at in this pic has been studied for decades, so it’s not in any doubt.