New preprint: Pumpy is here!

Together with the he lab of Ricardo Henriques, we’re finally ready to present #Pumpy to the world, in the form of a preprint on bioRxiv!

Pumpy (short for Pumpy McPumpface, official name NanoJ-Fluidics) is a pump array made in LEGO, controlled by an Arduino and open-source software (compatible with ImageJ/Fiji and Micro-Manager). NanoJ-Fluidics automates sample fluid exchange right on the microscope stage, allowing complex workflows using your standard chambers, tubing and reagents: live-to-fixed correlative imaging, sequential staining/imaging/washing protocols…

In one application,  we used NanoJ-Fluidics to visualize the dynamics of actin in living cells (using SRRF super-resolved processing), and to perform nanoscale imaging of actin using STORM on the same cell after online fixation and labeling with phalloidin.

 

We also used NanoJ-Fluidics for sequential multiplexed STORM/PAINT imaging, and we obtained 5-channel super-resolved images of actin, intermediate filaments, microtubules, clathrin and mitochondria in cells with minimal intervention during imaging.

 

If you are interested in making your own, head over to the NanoJ-Fluidics wiki where you will find everything:  LEGO parts, assembly instructions, control software and more!

We support PreLights, a preprint highlighting initiative

Today the Company of Biologists (a non-profit organization that published journals such as Development or the Journal of Cell Science) launched a new website called PreLights. PreLights is a website where a group of young researchers (the PreLighters) curate biology preprints, highlighting their value and importance. Some highlights also feature feedback from authors. We are happy to support this open science initiative by providing the PreLight banner image: neurons in culture that reflects the connections and interactions preLights wants to promote.

The SQUIRREL is out!

Our article with the Henriques and Mercer labs is now out in Nature Methods. Previously posted on bioRxiv, it proposes a new metric to measure the quality of super-resolution images called NanoJ-SQUIRREL (Super-resolution Quantitative Image Rating and Reporting of Error Locations). Simply put, it compares the image to a reference diffraction-limited image, allowing to detect  artefacts and missing features in the super-resolved image.

We used SQUIRREL to determine when to stop a STORM acquisition, showing that the longest acquisition was not always the best. In this case of imaging actin rings along axons labeled with phalloïdin, the quality of the STORM image as measured by SQUIRREL peaks after 30,000 frames and slightly drops when reaching 60,000 frames:

Another application of SQUIRREL is to optimize a DNA-PAINT experiment. In DNA-PAINT imaging, the density of the blinking events can be tuned by varying the concentration of the imager strand. We imaged clathrin-coated pits in a glial cell and the blinking sequence were processed with different algorithms. Each algorithm had a specific optimal blinking density (and thus imager strand concentration): SRRF was better with a denser blinking sequence, while pure localization algorithms such as MLE or CoM require a sparser acquisition sequence:

There are a lot more applications in the paper and its hefty supplementary file, go check it! Then try it thanks to the easy to use, open-source plugin for the ImageJ/Fiji plugin software.

Finally, this project is a striking example of open science in action. I (Christophe) met and started to collaborate with Ricardo and Jason on Twitter; the manuscript was posted on bioRxiv, where it got feedback from beta-testers and caught the attention of editors, before being accepted for publication six months later. Congrats to Siân, David, Caron, Pedro, and everyone!

 

New article out: myosin II at the axon initial segment

Our collaboration with the lab of Jim Salzer (NYU) is just out. We visualized the association of phospho-myosin light chain (pMLC, an activator of the contractile myosin-II) with actin rings along the axon initial segment (AIS). This was done using two-color STORM. Moreover, acute treatment with KCL (mimicking elevated activity) resulted in a disappearance of the phospho-MLC signal before the disorganization of the actin rings. This suggests that myosin-II contractility has a role in setting the AIS shape and position along the axon, and that release of this contractility could be a key remodelling step for the activity-dependant plasticity of the AIS.

Talks in Paris

Christophe gave two talks in Paris at the end of November, one at the Institut de la Vision in Paris, invited by Romain Brette for a PhD committee. Romain is studying how AIS position is regulated by neuronal morphology to optimize the electrogenic properties of each neuron, and he is interested by a lot of other things about the brain and its secrets as you can read on his blog.

The other talk was at a meeting at the meeting of the “Team Samples” from the RT-MFM CNRS technology group. There were interesting discussions on how to properly prepare samples for STORM and DNA-PAINT with the French super-resolution microscopy community.