New preprint: Get the best 3D localization microscopy with DAISY

We have a new preprint out! It’s a collaboration with the group of Sandrine Lévêque-Fort at ISMO (Orsay, France) based on the PhD work of Clément Cabriel. They previously used supercritical-angle fluorescence to measure the height of fluorophores at the proximity of the coverslip. This technique, called Direct Optical Nanoscopy with Axially Localized Detection (DONALD), could bring the resolution down to 15 nm for 3D localization microscopy. Now they have combined the SAF-based method with cylindrical lens astigmatism to obtain a robust and precise 3D localization of fluorophores over ~1.5 µm above the coverslip, retaining the key advantafges of DONALD: drift-free, tilt-insensitive and achromatic. The new technique, called Dual-view Astigmatic Imaging with SAF Yield (DAISY 😉), allowed to image in 3D the periodic scaffold of adducin and ß-spectrin along axons of cultured neurons, as you can see on the Figure below:
Cabriel_Preprint

Just published: our review on the architecture of axonal actin

Marie-Jeanne and Christophe wrote a review detailing how recent discoveries renewed the understanding of axonal actin organization. In the axon shaft itself, new nano-structures such as rings, hotspots and trails have been described, but their function remains to be elucidated. At presynapses, the precise architecture of actin is still elusive, and contradicting findings have been reported regarding its function. This is an exciting time to study actin in axons!

The review is now published in Molecular and Cellular Neuroscience, and will be part of a special issue on “Membrane Trafficking and Cytoskeletal Dynamics in Neuronal Function”. If you don’t have access to the review, a preprint manuscript is available on Zenodo.

Fig.2 from Papandreou & Leterrier 2018

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.