SAFe 180 | Single-Molecule Localization Microscope / Add-On

Cell Biology

• Cytoskeleton
• Metabolism
• Membrane Proteins
• Transport
• Signaling

Bacteriology

• Antibiotic resistance
• Replication
• Membranes

Immunology

• Immune receptors

Neuroscience

• Axon nanoscale architecture

Nucleus

• Replication and transcription
• Chromatin
• Nuclear pores and nuclear envelope

SMLM capabilities: from structure to dynamics

Current SMLM approaches only differ in how the fluorophores activation-inactivation is induced. Among them, STORM, PALM and PAINT resolve spatial structures with nanometric precision, while SPT reveals temporal dynamic processes in living cells.

Structures …

dSTORM

direct STochastic Optical Reconstruction Microscopy

dSTORM exploits the photo switching properties of some fluorophores. In certain conditions, fluorophores can be sent to an intermediary state (the “dark state”) from which they randomly cycle between ON and OFF states.

• Standard organic fluorescent dyes (cyanines, rhodamines, oxazines…)
• Specific imaging buffer containing a reducer, which induces the transition to a dark state, and an oxygen scavenging system to stabilize this state before returning to the ground state.

PALM

PhotoActivated Localization Microscopy

PALM uses specific fluorescent fusion proteins called “photoactivatable” or “photoconvertible”. The ON state of these proteins can be randomly activated with the proper laser excitation.

• Photo-activatable or convertible fluorescent proteins (mEos3,2, Dendra2, PA-mCherry, mMaple,…);
• No specific buffer, live-cell compatible

PAINT

Point Accumulation for Imaging in Nanoscale Topography

PAINT uses specific dyes that are ON only when they are transiently bound to their target. W ith the appropriate dye concentrations, it is possible to achieve a regime where only a few dyes are ON at the same time. DNA-PAINT uses short DNA single strands coupled to fluorophores. These probes are ON when they transiently hybridize to the complementary DNA strand coupled to an antibody targeting the structure of interest.

• Specific fluorophores that have the ability to emit fluorescence only upon binding to their biological target (ex: Nile Red, which fluoresces only when interacting with membranes)
• No specific buffer, live-cell compatible.

Dynamics…

SPT: Single Particle Tracking

SPT in SMLM combines Single Particle Tracking with SMLM (PALM or STORM) to obtain spatially and temporally highly resolved diffusion maps of single molecules.SPT need a precise algorithm to found and draw tracks from each molecule in the cell, state of the art algorithm are implemented in Neo Softwares.

• Sellés, J., Penrad-Mobayed, M., Guillaume, C. et al. Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy. Sci Rep 7, 14732 (2017). https://doi.org/10.1038/s41598-017-15433-2
• Rym Boudjemaa , Clément Cabriel , Florence Dubois-Brissonnet et al. Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus.  ASM Journals (2018) https://doi.org/10.1128/AAC.00023-18 
• Anaïs Bouissou, Amsha Proag, Nicolas Bourg, et  al. Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring ACS Nano 2017 11 (4), 4028-4040 DOI: 10.1021/acsnano.7b00622
• Cabriel, C., Bourg, N., Jouchet, P. et al. Combining 3D single molecule localization strategies for reproducible bioimaging. Nat Commun 10, 1980 (2019). https://doi.org/10.1038/s41467-019-09901-8
• Clément Cabriel, Nicolas Bourg, Guillaume Dupuis, and Sandrine Lévêque-Fort, Aberration-accounting calibration for 3D single-molecule localization microscopy,  Opt. Lett. 43, 174-177 (2018) ​​https://doi.org/10.1364/OL.43.000174
• Clément Cabriel, Nicolas Bourg, Guillaume Dupuis, and Sandrine Lévêque-Fort, “Aberration-accounting calibration for 3D single-molecule localization microscopy,” Opt. Lett. 43, 174-177 (2018) https://doi.org/10.1364/OL.43.000174
• Béatrice Durel, Charles Kervrann, Giulia Bertolin. Quantitative dSTORM super-resolution microscopy localizes Aurora kinase A/AURKA in the mitochondrial matrix. bioRxiv 2020.11.20.390948 https://doi.org/10.1101/2020.11.20.390948
• Felix Schneider, Thuy-An Duong, Isabell Metz, Jannik Winkelmeier, Christian A. Hübner, Ulrike Endesfelder, Marco B. Rust, Mutual functional dependence of cyclase-associated protein 1 (CAP1) and cofilin1 in neuronal actin dynamics and growth cone function, Progress in Neurobiology, Volume 202, 2021,  102050, ISSN 0301-0082, https://doi.org/10.1016/j.pneurobio.2021.102050.
• Mau, A., Friedl, K., Leterrier, C. et al. Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields. Nat Commun 12, 3077 (2021). https://doi.org/10.1038/s41467-021-23405-4
• Quentin Szabo, Daniel Jost, Jia-Ming Chang, et al. TADs are 3D structural units of higher-order chromosome organization in Drosophila. SCIENCE ADVANCES 28 FEB 2018 : EAAR8082. DOI: 10.1126/sciadv.aar8082
• Hershko, E., Weiss, L.E., Michaeli, T., & Shechtman, Y. (2018). Multicolor localization microscopy by deep learning. arXiv: Optics. aXiv: 1807.01637
• Philippe Codron, Julien Cassereau, Patrick Vourc’h, et al (2018). Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions, Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 19:5-6, 446-456, DOI: 10.1080/21678421.2018.1431787
• Stephen L. Berger, Alejandra Leo-Macias, Stephanie Yuen et al. Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment. Neuron 97:3. 2018 https://doi.org/10.1016/j.neuron.2017.12.039
• Culley, S., Albrecht, D., Jacobs, C. et al. Quantitative mapping and minimization of super-resolution optical imaging artifacts. Nat Methods 15, 263–266 (2018). https://doi.org/10.1038/nmeth.4605