Pumpy is out! Perform advanced microscopy experiments thanks to NanoJ-Fluidics

The LEGO Pumpy (or more officially NanoJ-Fluidics) paper is out ! A joint venture with the Henriques lab, this details how to build a fully open-source multi-channel syringe pumps with LEGO and Arduino. We provide examples on how to use it directly on the microscope for complex imaging protocols: live-to-fixed correlative acquisitions, image-analysis triggered fixation, sequential imaging… Check the video we put together showing the possibilities:

In the lab, we used Pumpy to perform complex STORM/PAINT multiplexed acquisitions. Here it’s a 5-color imaging of actin, mitochondria, intermediate filaments, microtubules and clathrin. It’s made with 1 single-color STORM and two 2-color PAINT sequential acquisitions:

Imaging was a breeze thanks to Pumpy, so the main challenge was to optimize the fixation and immunolabeling for 5 distinct targets. Great work from Ghislaine Caillol and Fanny Boroni-Rueda! Check the full article here for more.

New preprint: tips and tricks for SMLM

We have a new preprint out! Want to do good super-resolution images? We have put together all our SMLM tips and tricks. This is a methods paper that describes our SMLM workflow, using benchmark samples such as microtubules and clathrin-coated pits.

Our SMLM workflow
Optimized labeling for cellular structures

A good image starts with a good sample… that’s why the first part is about how to our fixation and immunolabeling procedures. Then it’s “just” a matter of imaging and processing. Tips for acquiring 3D-STORM images, as well as multi-color DNA-PAINT. Congrats to Angélique and Karoline for their beautiful images. Check our preprint to know everything, and let us know what you think!

3D-STORM images of microtubules, clathrin and actin
Multicolor STORM/DNA-PAINT imaging of microtubules, clathrin and actin

Just out: overview of the NanoJ framework for open-source super-resolution

The Henriques lab and its collaborators have a new paper out in the Journal of Physics D: Applied Physics. This is an overview of the NanoJ framework they are developing for open-source super-resolution in ImageJ/Fiji. In includes SRRF, SQUIRREL, NanoJ-Fluidics aka Pumpy, but also utilities for drift correction, chromatic aberration registration and single-article averaging. Have a look at the accepted manuscript here!

Welcome to Karoline

Today we welcomed a new team member, Karoline Friedl. She will be in the lab thanks to a collaboration with French startup Abbelight, and will help develop and test new super-resolution modalities for our projects. Welcome Karoline!

New paper out: slow axonal transport of actin via hotspots and trails

Our latest work (previously on bioRxiv) is now published in the Journal of Cell Biology. We collaborated with the Roy lab to reveal a new mechanism of slow axonal transport, based on the previous discovery of actin hotspots and trails. Hotspots are static actin clusters that appear and disappear within minutes every 3-4 µm along the axon. They generate the assembly of trails, long actin filaments that polymerize along the axon and collapse within seconds. Our new article first shows that trails polymerize at their barbed ends, located at the surface of hotspots. Each trail is thus pushed away from the hotspot as as it grows, resulting in a net displacement of actin monomers after trail collapse. In addition, trails grow in both directions (anterograde and retrograde), but with a small bias toward the tip of the axon (58% anterograde vs 42% retrograde).

The combination of these two processes (displacement of actin by trails and anterograde bias) results in the slow progress of actin along the axon. Modeling from the Jung lab allowed to determine the overall actin transport speed resulting from the hotspots and trails dynamics. Strikingly, this slow anterograde transport speed of actin (0.4 mm/day) precisely matches the values obtained by classic radio-labeling studies. This is a fundamentally new mechanism of slow axonal transport for cytoskeletal components, based on a biased assembly/disassembly mechanism rather than processive transport by motor proteins.

In this work, we used STORM imaging of axonal actin to pinpoint the architecture of hotspots, showing that the multi-directional growth of trails make them appear as asters when the axon is thicker (see Figure). Furthermore, we imaged hundreds of hotspots by STORM and quantified their diameter to ~200 nm. This is a first step toward elucidating the molecular organization of hotspots and trails, which will be crucial to understand their cellular functions.

Details of hotspots seen by STORM
Quantification of hotspots diameter from STORM images

MiFoBio 2018: the best microscopy workshop!

Christophe was lucky to spend a whole week at the “Microscopie Fonctionnelle en Biologie” aka MiFoBio workshop. Lots of fun attending dozens of cutting-edge workshops, trying super-resolution microscopes, discussing, DJing (!), and presenting the latest work from the lab.

More on Twitter: #MiFoBio2018

photo: @HenriquesLab
COS cell labeled for actin and microtubules, imaged using Gataca System’s LiveSR spinning disk microscope
COS cell labeled for actin and microtubules, imaged using Olympus’ SpinSR10 spinning disk microscope