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!
Christophe gave a conference on “The neuronal cytoskeleton revealed by cellular imaging” at the “Mechanobiology of polarized cell” summer school at the Ecole de Physique des Houches (in front of the Mont-Blanc!). This was organized by the PhD students of the Marie Curie Sklodowska International Training Network “Biochemical and mechanochemical signaling in polarized cells” (BIOPOL). A good occasion to present what super-resolution microscopy can reveal about polarized cells organization, and discuss about the mechanical implications of the axonal cytoskeleton architecture. See more from the #mechanopol18 hashtag on twitter.
Our image of a cultured neuron is displayed at the Montparnasse-Bienvenüe subway station in Paris! This is part of a 150 meter-long mural installed by the CNRS: the “Étonnant Vivant” exhibit, see more here (in French) and look at the entire mural here.
Christophe wrote a short review for the Journal of Neuroscience’s Viewpoints series, summarizing the latest results about the Axon Initial Segment (AIS). It’s out today in J. Neurosience latest issue. Also, a nice image of our neurons with their initial segments was chosen as the cover!
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.
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:
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!
Nikki van Bommel, a Master student from the University of Amsterdam (UvA), has joined the team for a 6-month internship, supported by an A*MIDEX fellowship. She previously worked with Joachim Goedhart (@joachimgoedhart) at UvA and wanted to learn how to do super-resolution microscopy. She will sure get plenty of training with us!
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.
Christophe was invited to the Weizmann Institute (Rehovot, Israel) for a Molecular Neuroscience Forum seminar. Beautiful Institute, lots of interesting discussions with neuroscientists and microscopists all around the Weizmann. Thanks Nicolas Panayotis and Mike Feinzilber for the invitation!