In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster

Schnorrenberg, Sebastian; Grotjohann, Tim; Vorbrüggen, Gerd; Herzig, Alf; Hell, Stefan W.; Jakobs, Stefan

doi:10.7554/elife.15567

Cited by 47 publications

(46 citation statements)

References 25 publications

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“…cytoskeletal transport | biophysics | kinesin | network | microtubules T he microtubule (MT) network in eukaryotic cells is typically a dense, highly variable, 3D mesh. MT network topologies are known to vary widely between cells (1) and even between cells of the same type and lineage (2). Within a network, MTs converge to form crossings at a variety of filament separations and angles of intersection (3).…”

mentioning

confidence: 99%

Cargo navigation across 3D microtubule intersections

Bergman

Bovyn

Doval

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The eukaryotic cell's microtubule cytoskeleton is a complex 3D filament network. Microtubules cross at a wide variety of separation distances and angles. Prior studies in vivo and in vitro suggest that cargo transport is affected by intersection geometry. However, geometric complexity is not yet widely appreciated as a regulatory factor in its own right, and mechanisms that underlie this mode of regulation are not well understood. We have used our recently reported 3D microtubule manipulation system to build filament crossings de novo in a purified in vitro environment and used them to assay kinesin-1-driven model cargo navigation. We found that 3D microtubule network geometry indeed significantly influences cargo routing, and in particular that it is possible to bias a cargo to pass or switch just by changing either filament spacing or angle. Furthermore, we captured our experimental results in a model which accounts for full 3D geometry, stochastic motion of the cargo and associated motors, as well as motor force production and force-dependent behavior. We used a combination of experimental and theoretical analysis to establish the detailed mechanisms underlying cargo navigation at microtubule crossings.

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mentioning

confidence: 99%

Cargo navigation across 3D microtubule intersections

Bergman

Bovyn

Doval

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…We recorded RESOLFT images of Drosophila melanogaster larvae body wall muscles expressing ubiquitously rsEGFP2 fused to α-tubulin as described in [23]. Tubulin is known to form helices with a diameter of approximately 25 nm, each turn comprising 13 dimers of αand β-tubulin and spaced 8 nm apart [24].…”

Section: Estimation Of the Mcf For Resolft Images Of Rsegfp2-α-tubulinmentioning

confidence: 99%

Molecular contribution function in RESOLFT nanoscopy

et al. 2019

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The ultimate objective of a microscope of the highest resolution is to map the molecules of interest in the sample. Traditionally, linear imaging systems are characterized by their spatial frequency transfer function, which is given, in real space, by the point spread function (PSF). By extending the concept of the PSF towards the molecular contribution function (MCF), that quantifies the average contribution of a single fluorophore to the image, a straightforward concept for counting fluorophores is obtained. Using reversible saturable optical fluorescence transitions (RESOLFT), fluorophores are effectively activated only in a small, subdiffraction-sized volume before they are read out. During readout the signal exhibits an increased variance due to the stochastic nature of prior activation, which scales quadratically with the brightness of the active fluorophores while the mean of the signal scales only linearly with it. Using a two-state Markov model for the activation, showing comparable behavior to the switching kinetics of the switchable fluorescent protein rsEGFP2, we can approximate quantitatively the MCF of RESOLFT nanoscopy allowing to count the number of fluorophores within a subdiffractionsized region of the sample. The method is validated on measurements of tubulin structures in Drosophila melagonaster larvae. Modeling and estimation of the MCF is a promising approach to quantitative microscopy.

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“…The general name of approaches overcoming the resolution limit is super-resolution uorescence microscopy. As an emerging method, the super-resolution reversible saturable optical linear (uorescence) transitions (RESOLFT) microscopy has to be mentioned, 83,84 as it is suitable for in vivo studies. 85 STED (stimulated emission depletion) and ground state depletion (GSD) microscopy rely on the working principle of RESOLFT.…”

Section: Super-resolution Techniquesmentioning

confidence: 99%